Begin manual download - The Plant Cell

This article is published in The Plant Cell Online, The Plant Cell Preview Section, which publishes manuscripts accepted for publication after they
have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces
normal time to publication by several weeks.
Proteomic Analysis of the Eyespot of Chlamydomonas
reinhardtii Provides Novel Insights into Its Components
and Tactic Movements W
Melanie Schmidt, a,1 Gunther Geßner, b,1 Matthias Luff, a,1 Ines Heiland, b Volker Wagner, b Marc Kaminski, b
Stefan Geimer, c Nicole Eitzinger, a Tobias Reißenweber, a Olga Voytsekh, b Monika Fiedler, b Maria Mittag, b
and Georg Kreimer a,2
a Institute of Biology, Friedrich-Alexander-University, D-91058 Erlangen, Germany
b Institute of General Botany and Plant Physiology, Friedrich-Schiller-University, D-07743 Jena, Germany
c Cell Biology/Electron Microscopy, University of Bayreuth, D-95440 Bayreuth, Germany
Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further
understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and
the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure
to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the
identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into
structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling
pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have
performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein
kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light.
We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that
silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.
INTRODUCTION
Flagellate green algae can perceive light information via a primitive
visual system, the eyespot apparatus. Light causes two
major types of behavioral responses in these algae. One is
phototaxis, the directed swimming toward or away from the light
source. The other, photoshock, is observed when the cells experience
a large and sudden change in light intensity. In most green
algae, the photoshock response is accompanied by a transient
stop in movement, followed by a short period of backward
swimming, after which normal forward swimming in a random
direction is resumed. So far, only a few molecular signaling components
of these two behavioral responses to light are known.
Both involve transmembrane Ca 2þ fluxes, which finally lead to
temporary changes in flagellar beating. In addition, excitation of
rhodopsins located in the eyespot apparatus initiates a cascade
of rapid electrical responses finally leading to changes in flagellar
beating and peculiar photoresponses (reviewed in Nultsch, 1975;
Witman, 1993; Kreimer, 2001; Sineshchekov and Govorunova,
2001; Kateriya et al., 2004).
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed. E-mail gkreimer@
biologie.uni-erlangen.de; fax 49-09131-8528215.
The authors responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) are: Maria Mittag (m.mittag@
uni-jena.de) and Georg Kreimer (gkreimer@biologie.uni-erlangen.de).
W Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.106.041749.
In the light microscope, the eyespot is seen peripherally near
the cell’s equator as a conspicuous, singular orange-red spot
(Figure 1A). The ultrastructure of the functional eyespot apparatus
is complex and involves local specializations of membranes
from different compartments (reviewed in Melkonian and Robenek,
1984; Kreimer, 2001). In the green model alga Chlamydomonas
reinhardtii, the eyespot apparatus is usually composed of two
highly ordered layers of carotenoid-rich lipid globules inside the
chloroplast (Figures 1B and 1C). The globules exhibit a remarkably
constant diameter of 80 to 130 nm and are subtended by a
thylakoid membrane. Additionally, the outermost globule layer is
attached to specialized areas of the two chloroplast envelope
membranes and the adjacent plasma membrane (Figures 1B and
1C). The plasma membrane and the outer chloroplast envelope
membrane above the eyespot globules are extremely rich in intramembrane
particles resembling most likely membrane proteins
(Melkonian and Robenek, 1984).
The photoreceptors identified so far are generally believed to
be located in this plasma membrane patch. Phototaxis requires
the cell to determine the direction of incident light. C. reinhardtii
most likely accomplishes this by monitoring the modulation of
the light intensity reaching its photoreceptors as the cell rolls
around its longitudinal cell axis during helical forward swimming.
The eyespot globule layers are important for modulation of the
light intensity. They confer increased directionality and contrast
to the photoreceptors by a dual action. First, they shield them
from light passing through the cell body. Second, they reflect
light falling directly on the eyespot that is not absorbed by the
photoreceptors back onto the overlying plasma membrane.
The Plant Cell Preview, www.aspb.org ª 2006 American Society of Plant Biologists 1 of 23

2 of 23 The Plant Cell
Figure 1. Eyespot Location, Structure, and Isolation of a Fraction
Enriched in Eyespot Apparatuses by Sucrose Gradient Centrifugation.
(A) Differential interference contrast image of a living cell. The arrow indicates
the position of the carotenoid-rich eyespot apparatus. Bar ¼ 10 mm.
(B) Schematic drawing of the eyespot apparatus of C. reinhardtii illustrating
the different components of this complex light sensor. Asterisks
indicate the carotenoid-rich eyespot globule layers inside the chloroplast,
which are associated with thylakoids (arrowheads). The outermost
layer is associated with the chloroplast envelope (large arrows). The
plasma membrane (small arrow) is closely attached to the chloroplast
envelope in the region overlying the eyespot globule layers. In addition,
the plasma membrane and the outer chloroplast envelope are enriched in
intramembrane particles in this area.
(C) Transmission electron micrograph of the eyespot apparatus of
C. reinhardtii. Labeling was done according to (B). Bar ¼ 300 nm.
(D) Distribution of the fraction enriched in eyespot apparatuses
(brackets) after flotation on discontinuous sucrose gradients. 1, separation
of the cell homogenate; 2, separation of the fraction after the first
purification step; 3, separation of the fraction after the second purification
step.
Thus, reflection amplifies the light signal at the photoreceptor
location and thereby increases their excitation probability (Foster
and Smyth, 1980; Kreimer and Melkonian, 1990; Kreimer et al.,
1992; Witman, 1993). Both absorption and reflection increase the
front-to-back contrast at the location of the photoreceptors up to
eightfold (Harz et al., 1992). In addition, the optical properties of
the eyespot apparatus and thereby the generated signal are
influenced by the swimming direction relative to the light source
(Hegemann and Harz, 1998). Briefly, the signal received by the
eyespot apparatus is low and nearly constant when the swimming
direction of the cells is well aligned with the light direction
but changes when swimming direction deviates from light direction.
This periodic signal is then processed in an as yet unknown
way and finally initiates corrective flagellar responses to realign
the swimming path. Thus, the whole complex (i.e., the special-
ized membranes and the eyespot globules forming the functional
eyespot apparatus) is important for optimal performance of this
primitive visual system. This has been demonstrated by analysis
of mutants defective in the formation of the eyespot globule
layers (Morel-Laurens and Feinleib, 1983; Kreimer et al., 1992).
Due to the elaborate structures of algal eyespot apparatuses and
the known presence of rhodopsins in some lineages, algae are
thought to play an important role in the evolution of photoreception
and eyes (Gehring, 2004). Therefore, the structural components
forming this early visual system and the signaling cascade
from the photoreceptor(s) to tactic movements are not only of
great interest to plant biologists but also for developmental and
other biologists. This is highlighted by the fact that one of the
Figure 2. Characterization of the Final Fraction Enriched in Eyespot
Apparatus Fragments by Transmission Electron Microscopy.
(A) to (C) Whole-mount preparations. Overview (A); details ([B] and [C]).
Note that the eyespot fragments tend to aggregate. Bars ¼ 2500 nm (A)
and 400 nm ([B] and [C]).
(D) to (H) Thin sections. White arrow heads indicate the contact sites
between the eyespot globules. Black arrowheads indicate eyespot
membranes, partially associated with fuzzy fibrilar material typically
observed in situ between the plasma membrane and chloroplast envelope
in the region of the eyespot apparatus. Bars ¼ 250 nm ([D], [E], [G],
[H], and[F]) and 150 nm (F).

hodopsin-like photoreceptors of C. reinhardtii can light-stimulate
neurons and trigger behavioral responses in Caenorhabditis
elegans (Boyden et al., 2005; Nagel et al., 2003, 2005).
In C. reinhardtii, several mutations affecting eyespot assembly
and positioning are known (Hartshorne, 1953; Morel-Laurens and
Feinleib, 1983; Pazour et al., 1995; Lamb et al., 1999; Nakamura
et al., 2001; Roberts et al., 2001). Five loci solely involved in
formation and/or correct positioning of the eyespot apparatus
have been identified so far. The mutant approach has recently led
to identification of two genes (min1 and eye2) that are involved in
eyespot assembly (Roberts et al., 2001; Dieckmann, 2003). In
min1 mutant strains, only miniature eyespots are formed,
whereas mutations in eye2 induce loss of a visible eyespot.
However, individual eyespot globules are still detectable by
electron microscopy in the mutant eye2 (Lamb et al., 1999).
Thus, general formation of the globules is probably not affected.
The eye2 gene product belongs to the thioredoxin superfamily
and exhibits no overall homology to any protein in the databases.
EYE2 might act as a specific chaperone in eyespot assembly. The
min1 gene also encodes a protein with little homology to known
proteins (Dieckmann, 2003). In addition to these proteins important
for eyespot development and size control, only four proteins
related to function of the eyespot apparatus have been identified
so far at the molecular level. These are the two unique seventransmembrane
domain photoreceptors COP3 and COP4, which
both act as directly light-gated ion channels (Nagel et al., 2002,
2003; Sineshchekov et al., 2002; Suzuki et al., 2003; Govorunova
et al., 2004). It should be noted that the same proteins have been
named differently by independent research groups (see Table
1 for the different nomenclature). COP3 and COP4 can initiate
Chlamydomonas Eyespot Proteome 3 of 23
Figure 3. Spectral Analysis and SDS-PAGE Analysis Demonstrates That Thylakoids Are Not Dominant in the Fraction of Eyespot Fragments.
(A) Normalized absorption spectra of the final fraction in aqueous solution (dashed line) and in 90% acetone (solid line).
(B) Comparison of the protein pattern of the eyespot fraction and isolated thylakoids. Proteins were separated on SDS-PAGE and stained with
Coomassie blue. The positions of molecular mass markers are indicated on the right (in kilodaltons).
extremely fast depolarizations. Consequently, a truncated COP4,
which is permeable to monovalent and divalent cations (Nagel
et al., 2003), has recently been expressed in mammalian neurons
and used for their light stimulation (Boyden et al., 2005) as already
indicated above. In addition, two splicing variants of the abundant
retinal binding protein COP (COP1 and COP2) were identified
(Deininger et al., 1995; Fuhrmann et al., 2003). Although
original experiments suggested these proteins as photoreceptors
(Deininger et al., 1995), their silencing showed that they are
not acting as photoreceptors in phototaxis and photoshock
(Fuhrmann et al., 2001). Based on conserved domain structures,
further putative retinal binding proteins encoded in the genome of
C. reinhardtii have recently been postulated to be also involved in
phototaxis (Kateriya et al., 2004), but in these cases a functional
proof is still missing. In conclusion, only six proteins clearly
related to the functional eyespot apparatus have been identified
so far at the molecular level. Therefore, in this study, we intended
to purify the eyespot apparatus in its entire complexity (i.e., the
eyespot globules along with the specialized areas of the plasma
membrane, chloroplast envelope, and thylakoid membranes;
Figures 1B and 1C) to obtain a complete set of proteins from this
complex cell organelle by a proteomic approach, although some
loss of soluble proteins cannot be ruled out.
Notably, tactic (photo- and chemo-) movements in C. reinhardtii
are not only controlled by light but also by the circadian
clock (Bruce, 1970; Mergenhagen, 1984; Byrne et al., 1992). Both
the rhythms of phototaxis and chemotaxis can be entrained by
an LD cycle and continue under constant conditions of light (or
darkness) and temperature with a period of ;24 h. While
circadian phototaxis peaks during subjective day (Bruce, 1970;

4 of 23 The Plant Cell
Mergenhagen, 1984), the chemotactic response to ammonium
reaches its maximum during subjective night (Byrne et al., 1992).
Since the phase of circadian phototaxis can be reset by both red
and blue light (Johnson et al., 1991; Kondo et al., 1991), an
involvement of different photoreceptors in affecting circadian
rhythms is apparent. It can be expected that the eyespot apparatus
is involved in the entrainment of the endogenous clock via
photoreceptors and a connected signaling cascade, and it might
even contain key components of the endogenous oscillator itself.
In all eukaryotic model organisms studied so far, including Neurospora
crassa, Arabidopsis thaliana, Drosophila melanogaster,
mice, and humans, phosphorylation plays a key regulatory role
within the circadian oscillator. Crucial components of the endogenous
oscillator that are regulated via positive and negative
feedback loops are progressively phosphorylated, which supports
their interaction with other proteins, promotes their nuclear
entry, and finally leads to their degradation at a specific stage of
the circadian cycle (reviewed in Dunlap, 1999; Harmer et al.,
2001; Panda et al., 2002; Reppert and Weaver, 2002; Dunlap and
Loros, 2004). While the essential components of the circadian
oscillator in the above-mentioned model organisms are not
conserved in C. reinhardtii, the involved Ser-Thr kinases (casein
kinase 1 [CK1], CK2, and SHAGGY) and the Ser-Thr protein
phosphatases (PP2A and PP1) are highly conserved (Mittag et al.,
2005). Therefore, appearance of any of these proteins within the
eyespot proteome will be indicative of their potential function
toward circadian regulation of tactic movements.
Proteomics, which often involves gel electrophoresis,
nano-liquid chromatography in combination with electrospray
ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS),
and bioinformatic analysis, has become a powerful tool for the
investigation of proteins (Reinders et al., 2004). In plant model
organisms like C. reinhardtii and Arabidopsis, several large-scale
proteome analyses have been performed in recent years, resulting
in the characterization of cellular subfractions such as cilia (Pazour
et al., 2005), centrioles (Keller et al., 2005), the vegetative vacuole
(Carter et al., 2004), specific chloroplast subproteomes (Peltier
et al., 2002; Yamaguchi et al., 2002; Majeran et al., 2005), or the
phosphoproteome (Wagner et al., 2006). In this study, we have
characterized the proteome of the eyespot apparatus from
C. reinhardtii by 1365 peptides belonging to 583 different proteins.
In total, 202 proteins were identified via at least two peptides. Here,
we describe a detailed analysis of these 202 proteins, including
their possible roles in eyespot structure, development, specific
metabolic processes, and in tactic (photo- and chemo-) signaling
pathways. A functional analysis was performed with one of them,
CK1. Our results suggest that it is a major player in several
processes, including hatching, flagellum formation, and circadian
control of phototactic behavior.
RESULTS AND DISCUSSION
Isolation and Characterization of a Fraction Enriched in
Eyespot Apparatuses
One major objective of this study was to yield a strongly enriched
eyespot fraction to gain a rather complete proteome of this cell
organelle and to minimize contaminating proteins. Due to its
complex ultrastructure involving local specializations of different
compartments (Figures 1B and 1C), biochemical isolation of the
eyespot apparatus is a challenging task. Taking advantage of a
previously described method for the isolation of eyespot globules
from the green alga Spermatozopsis similis (Renninger et al.,
2001), we developed a procedure for the purification of the
C. reinhardtii eyespot apparatus that is based on flotation on
successive sucrose gradients (see Methods).
As a first visual marker for enrichment of eyespot apparatuses
during the purification procedure, we took the striking orangered
color of the eyespot globules (Figure 1A). A deep orange-red
fraction was separated from a weak yellow-orange fraction, the
bulk of chloroplasts, and cell debris by the first gradient centrifugation
step (Figure 1D). This carotenoid-rich fraction was then
purified by two additional gradient centrifugation steps to minimize
contamination by other cell organelles, thylakoids, and
soluble proteins and finally concentrated by a floating centrifugation
step (Figure 1D).
To verify enrichment of eyespot apparatuses and judge their
purity, the final fractions of three independent isolations were
analyzed by transmission electron microscopy (Figure 2). Wholemount
preparations revealed enrichment of globule aggregates
overlaid by membrane patches of varying size, which had a
strong tendency to form larger aggregates (Figures 2A to 2C). A
significant number of isolated single globules were not observed.
The average diameter of the individual globule aggregates (0.66
mm) was similar to that of the eyespot apparatus of C. reinhardtii
in situ (0.65 mm; Kreimer, 2001; Dieckmann, 2003). Thin sections
confirmed the successful isolation of fragments of eyespot apparatuses,
(i.e., globules associated with varying layers of membranes;
Figures 2D to 2H). The close association and arrangement
of these membrane patches with the eyespot globules strongly
suggests that they represent those parts of the plasma membrane,
the two chloroplast envelope membranes as well as the thylakoid
system that form, in conjunction with the eyespot globules, the
functional green algal eyespot apparatus (Figures 1B and 1C;
Foster and Smyth, 1980; Melkonian and Robenek, 1984; Kreimer,
1994). The structural preservation of the isolated eyespot fragments
varied. Although often close packing of the eyespot globules
was preserved and the diameter of many globules matched
the in vivo range of 80 to 130 nm (Kreimer, 2001), many globules
appeared to be fused during the isolation procedure and preparation
for electron microscopy (diameters > 250 nm). Except the
small amounts of membrane patches that were not associated
with eyespot globules (Figure 2A), no contaminations (e.g., by cell
debris, intact cell organelles, or flagella) were evident.
Spectral analysis of the pigments that are present in the
eyespot fraction was performed to verify that carotenoids were
present at a significantly higher rate in comparison with chlorophyll,
which is solely present within the thylakoid membranes
(Figure 1B). In aqueous solution, the main absorption peak was
observed in the yellow region of the spectrum (539.8 nm 6 1.7 nm;
Figure 3A). It coincides well with the peak observed for in vivo
eyespot reflectivity in C. reinhardtii (540 to 550 nm; Schaller and
Uhl, 1997). In acetone extracts, a typical carotenoid spectrum
with major absorption peaks at 449.5 nm 6 0.6 nm and 474.5 nm
6 1.3 nm was observed. Only a rather small chlorophyll peak at
680 nm was detectable (Figure 3A). The carotenoid:chlorophyll

Table 1. Functional Categorization and Characterization of Identified Proteins from the Eyespot Apparatus
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
Chlamydomonas Eyespot Proteome 5 of 23
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp TMDs b
Proteins important for eyespot development
C_490073 c /188648 10 EYE2, no eyespot þ
C_630002 c /156645 7 MIN1, mini-eyespot þ
Photoreceptors
C_500071c /95849 8 COP3/CHOP1/CSRA/Acop1; retinal
binding protein
þ
C_3230005c /164843d 5 COP1 and COP2; retinal binding protein ÿ
C_390092c /182032 3 COP4/CHOP2/CSRB/Acop2; retinal
binding protein
þ
C_120056/183965 3 PHOT; blue light photoreceptor ÿ
Phosphatases e
C_760036/193906 f 37 Protein with PP2Cc (Ser-Thr phosphatases) domain (þ)
C_760032/178366 g 22 Protein with PP2Cc (Ser-Thr phosphatases) domain (þ)
Kinasese C_60149/131695 12 Cyclic nucleotide-dependent protein
kinase II
ÿ
C_230061/113847g 10 Similar to proteins with AarF (predicted unusual protein
kinase) domain
(þ)
C_110160/192323d 3 Similar to proteins with AarF (predicted unusual protein
kinase) domain
(þ)
C_70149/137286 2 CK1 ÿ
Calcium-sensing and binding proteins
C_1010018/194676 8 Calcium sensing receptor (þ)
C_20012/186813 5 Protein with EF-hand, calcium binding motif h (þ)
C_280062/183554 4 Protein with EF-hand, calcium binding motif þ
C_20380/111945 f 2 Protein with EF-hand, calcium binding motif þ
C_40075/189454 f 2 Protein with FRQ1 (Ca 2þ binding protein) domain h (þ)
Putative chemotaxis-related proteins
C_1250029/189928 5 Similar to MCP (Nostoc punctiforme PCC 73102) h (þ)
C_390049/133829 4 Similar to UbiE/Coq5 methyltransferases ÿ
C_290078/149419
Channels
3 Putative methlytransferase (Thermosynechococcus
elongatus BP-1)
ÿ
C_280032/146232 8 Similar to voltage-dependent anion channel protein (þ)
C_2200010/127172 6 Porin-like protein (þ)
Retinal biosynthesis and retina-related proteins
C_970031/153728d 14 Similar to SOUL heme-binding proteins ÿ
C_80229/174292g 12 Similar to cyanobacterial retinal pigment epithelial
membrane protein and
lignostilbene-a,b-dioxygenase
(þ)
C_2440006/191453f 2 Similar to retinol dehydrogenase 13 (all-trans and 9-cis) (þ)
Membrane-associated/structural proteins
Proteins with PAP-fibrillin domain
C_500037/121152 g 16 Protein with PAP-fibrillin domain (þ)
C_2690006/176214 g 12 Protein with PAP-fibrillin domain h (þ)
C_30242 i 8 Protein with PAP-fibrillin domain ÿ
C_500033/193429 g 7 Protein with PAP-fibrillin domain (þ)
C_250022/190008 4 Protein with PAP-fibrillin domain ÿ
C_2460003/154241 3 Similar to harpin binding protein 1 (þ)
C_370103/169629 d 3 Protein with PAP-fibrillin domain (þ)
C_13870001/176214 g 2 Protein with PAP-fibrillin domain h (þ)
(Continued)

6 of 23 The Plant Cell
Table 1. (continued).
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp TMDs b
Others
C_840016/154677 3 Similar to algal-cell adhesin molecule, contains two
FAS1 domains
(þ)
C_190173i 2 Similar to Morn repeat protein 1 (þ)
Carotenoid and chlorophyll biosynthesis
C_1950004/194594 f 16 DXS, 1-deoxy-D-xylulose-5-phosphate synthase (þ)
C_230123/113656 d 7 Cyanobacterial z-carotene desaturase-like protein ÿ
C_1330031/195952 6 DVR, 3,8-divinyl protochlorophyllide a 8-vinyl reductase ÿ
C_180047/136810 4 GGR, geranylgeranyl reductase ÿ
C_280053/136589 4 POR, protochlorophyllide reductase (þ)
C_490019/78128 2 PDS, phytoene desaturase þ
C_330078/191043 d 2 PPX, protoporphyrinogen oxidase precursor (þ)
Lipid metabolism
C_570065/77062 13 Betaine lipid synthase, extraplastidic (þ)
C_6260003/113915j 3 Similar to long-chain acyl-CoA synthetases 2
(Arabidopsis)
ÿ
C_2030015/98450f 3 Similar to proteins with PlsC domain
(1-acyl-sn-glycerol-3-phosphate acyltransferase)
þ
C_280073i 3 Similar to proteins with a diacylglycerol acyltransferase
domain
(þ)
C_220002/119132d 3 Similar to cyclopropane fatty acid synthases ÿ
C_7940001/113915j 2 Similar to a putative acyl-CoA synthetase (Oryza sativa) ÿ
C_100060/116066d Chloroplast ATP synthase
2 Similar to 3-b hydroxysteroid dehydrogenase/
isomerase (Anabaena variabilis ATCC 29413)
ÿ
Cp genome 21 CF1 ATP synthase b-subunit (þ)
Cp genome 20 CF1 ATP synthase a-subunit ÿ
C_17110002k 14 CF1 ATP synthase, a-subunit ÿ
Cp genome 8 CF0 ATP synthase subunit I þ
C_200074/134235 7 CF1 ATP synthase, g-chain ÿ
C_1610012/132678d 5 CF1 ATP synthase, d-chain ÿ
C_28050002k 4 CF1 ATP synthase e-subunit ÿ
Cp genome 4 CF1 ATP synthase e-subunit ÿ
C_480050k /105641k 2 CF1 ATP synthase, b-subunit (þ)
Cp genome 2 CF0 ATP synthase subunit IV þ
Photosystem II and related proteins
C_880018/148057d 10 PSBP oxygen-evolving enhancer protein 2 (23-kD
subunit of oxygen evolving complex
of photosystem II)
(þ)
Cp genome 9 Photosystem II P680 chlorophyll A apoprotein þ
C_940002/130316 7 PSBO oxygen-evolving enhancer protein 1 (33-kD
subunit of oxygen evolving complex
of photosystem II)
(þ)
C_32080002k 6 Photosystem II P680 chlorophyll A apoprotein þ
Cp genome 6 Photosystem II 44-kD reaction center protein þ
C_1340006/153656d 5 PsbQ, oxygen evolving enhancer protein 3 (þ)
Cp genome 4 Photosystem II reaction center protein D2 þ
Cp genome 3 Photosystem II reaction center protein D1 þ
C_270022/112806f 3 HCF136, photosystem II stability/assembly factor (þ)
C_180041/190151 2 Putative lumen protein, related to OEE3, PsbQ þ
C_770034/193552 2 Lhc-like protein Lhl3 (þ)
Cp genome 2 Photosystem II reaction center protein H þ
LHCII proteins
C_10030/184810 7 Lhcb4, minor chlorophyll a/b binding protein
of photosystem II
(þ)
(Continued)

Table 1. (continued).
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
Chlamydomonas Eyespot Proteome 7 of 23
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp TMDs b
C_530002/130414 6 Lhcb5, minor chlorophyll a/b binding protein
of photosystem II
(þ)
C_110177/131156 4 LhcbM5, chlorophyll a/b binding protein of LHCII (þ)
C_1460005/138110 4 LhcbM9, chlorophyll a/b binding protein of LHCII (þ)
C_70041/184070 3 LhcbM7, chlorophyll a/b binding protein of LHCII (þ)
C_1190021/178631d 3 LhcbM1, chlorophyll a/b binding protein of LHCII (þ)
C_2050001/186064 2 LhcbM3, chlorophyll a/b binding protein of LHCII (þ)
Cytochrome b6f complex and plastocyanin
Cp genome 17 Cytochrome f þ
C_1090006/185971 7 PETO, cytochrome b6f–associated phosphoprotein
(þ)
C_20090002k 4 Cytochrome b6 þ
Cp genome 4 Cytochrome b6 þ
C_1580045/108310f 2 Plastocyanin (þ)
Photosystem I and related proteins
C_100097/136252 12 Crd1, copper response defect 1 protein ÿ
C_1940014/130914 5 PSAF, photosystem I reaction center subunit III (þ)
C_300013/120177 5 PSAD, photosystem I reaction center subunit II ÿ
C_450050/182959 3 PSAH, photosystem I reaction center subunit VI (þ)
C_490082/128002 3 Cth1, copper target homolog 1 protein ÿ
C_1220023/165418 2 PSAG, photosystem I reaction center subunit V (þ)
C_50019/133651 2 PSAN, photosystem I reaction centre subunit N þ
Cp genome 2 Photosystem I assembly protein ycf4 þ
Cp genome 2 Photosystem I P700 chlorophyll A apoprotein A2 þ
LHCI proteins
C_270001/134203 7 Lhca8, light-harvesting protein of photosystem I (þ)
C_100004/78552 6 Lhca7, light-harvesting protein of photosystem I (þ)
C_1460019/174723 4 Lhca1, light-harvesting protein of photosystem I ÿ
C_1610027/153678 4 Lhca3, light-harvesting protein of photosystem I (þ)
C_490067/183029 4 Lhca2, light-harvesting protein of photosystem I (þ)
C_130138/133575 2 Lhca5, light-harvesting protein of photosystem I ÿ
Ferredoxin and thioredoxin-related proteins
C_680071/182093 3 Related to 2Fe2S ferredoxin ÿ
C_200197/142363 2 PRX1 2-cys peroxiredoxin, chloroplast ÿ
Protein translocation, assembly and
chaperones, chloroplast
C_750041/126835 14 Heat Shock Protein 70B ÿ
C_390061/133800 7 Protein disulfide isomerase 1, RB60 (þ)
C_270042/187077 d 6 Similar to Tic62 (þ)
C_10066/173082 d 4 Similar to Tic22 ÿ
C_10196/187295 d 3 Albino 3-like protein (þ)
C_30247/100945 d 3 Putative peptidyl-prolyl cis-trans isomerase ÿ
C_460094/143879 2 Similar to TOC75 ÿ
C_1110032/172529 g 2 Similar to TOC90 ÿ
C_490015/55286 d 2 Chloroplast DnaJ-like protein 1 ÿ
Diverse chloroplast envelope proteins
C_320089/143003 3 Similar to putative chloroplast inner envelope protein
(O. sativa)
ÿ
C_2350003/195230 2 Plastidic ATP/ADP transporter þ
Stromal proteins
C_280107/129019 9 GapA, glyceraldehyde-3-phosphate dehydrogenase A ÿ
C_30013/190455 5 Malate dehydrogenase, sodium acetate induced (þ)
C_30202/101042 f 3 SBPase, sedoheptulose-bisphosphatase ÿ
C_4220001/82495 d 3 FBPase, fructose-1,6-bisphosphate aldolase ÿ
(Continued)

8 of 23 The Plant Cell
Table 1. (continued).
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp TMDs b
C_950008/184105 2 PRK, phosphoribulokinase ÿ
Proteases, peptidases, and related proteins
Chloroplast
C_1620016/114776d 12 FtsH protease 1, probably chloroplast targeted þ
C_10225/175878f 6 FtsH protease 2, probable ortholog of Arabidopsis
FtsH2
þ
C_100122/101349f 5 Putative chloroplast thylakoidal processing peptidase (þ)
C_4010001/140380d Others
2 Similar to ClpC or ClpD chaperone, Hsp100 family,
ATP-dependent subunit of Clp protease
ÿ
C_240088/116429g Cytosolic proteins
7 Similar to metalloendopeptidases (þ)
C_1340012/185673 6 HSP70a, Heat Shock Protein 70 a, light
and heat inducible
(þ)
C_550067/158129d 4 MDH, cytosolic malate dehydrogenase þ
C_1460023/191668d 3 Isocitrate lyase, cytosolic or peroxisomal ÿ
C_970001/107783d Mitochondrial
2 Similar to expressed protein with saccharopine
dehydrogenase domain
(þ)
C_710028/192142 11 ASA2, putative mitochondrial ATP
syntase-associated protein
(þ)
C_3890001/78348 8 b-Subunit of mitochondrial ATP synthase ÿ
C_730039/138185 6 ANT1, mitochondrial ADP/ATP translocator protein þ
C_420010/78831 5 ASA1, ATP syntase-associated protein 1
(P60 or MASAP)
ÿ
C_230150/182740 5 ASA4, putative mitochondrial ATP syntase-associated
protein
ÿ
C_490077/100068 4 Putative mitochondrial processing peptidase a-subunit (þ)
C_1540001/159938 3 Hypothetical mitochondrial carrier protein (þ)
C_530055/191516 3 Putative mitochondrial dicarboxylate transporter þ
C_20064/76602 2 a-Subunit of the mitochondrial ATP synthase (þ)
C_710036/192157 2 NUOP6, mitochondrial NADH:ubiquinone oxidoreductase
19-kD subunit precursor
ÿ
C_90043/195712d 2 Cytochrome c oxidase, subunit VIb/COX12 ÿ
C_750022/152682 2 ASA3, putative mitochondrial ATP syntase-associated
protein
ÿ
C_1380005/194183 2 ATPase, g-subunit, probably mitochondria targeted ÿ
C_260140/111351d 2 Mitochondrial import receptor subunit Tom40-like ÿ
Golgi/endoplasmic reticulum/vesicle trafficking
C_490107/94234d 4 Similar to Golgi apparatus protein 1 isoform 2
(Canis familiaris)
þ
C_50001/133859 3 Heat Shock Protein 70, endoplasmic reticulum isoform þ
C_250128/78954 2 Calreticulin (þ)
C_290072/118846f 2 CYN20-1, peptidyl-prolyl cis-trans isomerase
(rotamase), cyclophilin type
þ
C_490065/144604 2 Putative peptidase þ
C_10830001/186126 2 ADP-ribosylation factor-like protein ÿ
Cytoskeleton
C_1320004/186023 2 TUA2, a tubulin 2 (þ)
Ribosomes, translation, and DNA-related
C_2390008/195598d 5 RPL4, cytosolic ribosomal protein L4 (þ)
C_1060035/160406 3 Histone H2A ÿ
C_290113/104082f 3 Similar to histone-like bacterial DNA binding protein;
possible targeting to organelle
ÿ
C_2370012/195131d 3 RPL22, cytosolic ribosomal protein L22 ÿ
(Continued)

Table 1. (continued).
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
Chlamydomonas Eyespot Proteome 9 of 23
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp TMDs b
C_380026/105289 f 3 RPL7a, cytosolic ribosomal protein L7a ÿ
C_930034/168484 3 RPS3a, cytosolic ribosomal protein S3a ÿ
C_430028/105734 f 3 RACK1, component of cytosolic 40S subunit ÿ
C_870056/145271 3 RPL14, cytosolic ribosomal protein L14 ÿ
Cp genome 3 Chloroplast RNA polymerase b-subunit ÿ
C_3470003/24289 2 RPS15, cytosolic ribosomal protein S15 ÿ
C_1060004 l 2 HFO8/HFO22, histone H4 ÿ
C_3320003/129809 2 RPL13, cytosolic ribosomal protein L13 ÿ
C_130042/126059 2 RPSa, cytosolic ribosomal protein Sa ÿ
C_1060006/123665 2 HTR5, histone H3 ÿ
C_3670002/174711 g 2 RPL23a, cytosolic ribosomal protein L23a (þ)
C_380137/162845 2 RPL13a, cytosolic ribosomal protein L13a ÿ
C_1480038/143072 2 RPS24, cytosolic ribosomal protein S24 ÿ
C_480013/24344 2 RPS14, cytosolic ribosomal protein S14 ÿ
C_90190/188837 g 2 RPS4A, cytosolic ribosomal protein S4E ÿ
Others
C_540038/184617 8 Similar to zonadhesin (Mus musculus) ÿ
C_410057/116285f 8 Similar to MGC86418 protein (Xenopus laevis);
FAD_Synth, Riboflavin kinase/FAD
synthetase domains
ÿ
C_1400008/152568 8 Similar to UDP-N-acetylglucosamine
pyrophosphorylase-like proteins
(þ)
C_80056/184328 5 Similar to chitinase, contains two LysM domains þ
C_190016/122660d 2 Similar to aldo-keto reductase/oxidoreductase
(Arabidopsis)
(þ)
C_950022/103066f 2 Similar to riboflavin biosynthesis-related protein
(Arabidopsis); RibD, pyrimidine deaminase
(coenzyme metabolism) domain
ÿ
C_1040013/132437 2 Similar to putative NADPH-dependent reductase
(O. sativa japonica cultivar group)
(þ)
C_1330014/146801 2 Similar to formate acetyltransferase
(Thermosynechococcus elongatus BP-1);
PFL1, pyruvate formate lyase domain
ÿ
C_1490014/122298d 2 Similar to amidophosphoribosyl transferase
(Ralstonia metallidurans CH34); PurF domain
ÿ
C_1690020/99287, 113924, 54929m 2 Similar to adenylosuccinate lyase
(Pseudomonas syringae pv Phaseolicola)
ÿ
C_4220002/153473 2 Similar to amidophosphoribosyl transferase
(Silicibacter sp TM1040); Gln amidotransferases
class-II (GN-AT) GPAT-type domain
ÿ
C_2020016/154307
Conserved proteins of yet unknown function
2 Similar to methyltetrahydropteroyltriglutamatehomocysteine
methyltransferase (Met synthase,
vitamin B12-independent isozyme)
(þ)
C_1670026/121991g 11 Similar to conserved plant/cyanobacterial proteins
of unknown functions, contains
two DUF1350 domains
(þ)
Cp genome 7 Hypothetical protein ChreCp059 þ
C_350047/183568d 6 Similar to conserved hypothetical protein
(Prochlorococcus marinus strain NATL2A)
ÿ
C_1550001/145347 6 Similar to hypothetical protein TeryDRAFT_4244
(Trichodesmium erythraeum IMS101)
ÿ
C_1580021/60278 5 Similar to DUF477 domain containing
proteins
þ
C_140123/160137d 5 Similar to DUF393 domain containing
protein of unknown function (Crocosphaera
watsonii WH 8501) contains to two CBS domains
(þ)
(Continued)

10 of 23 The Plant Cell
Table 1. (continued).
Gene Model (JGI Version 2)/Protein ID (JGI
Version 3) or cp a
No. of Different
Peptides
ratio (absorbance 478 nm:680 nm) ranged between 60 and 70 in
different preparations (Figure 3A). A comparison of the amount of
chlorophyll present in the crude extract with the amount of
chlorophyll that could be found in the final eyespot fraction
revealed that

was largely reduced. It was shown with freeze fracturing that
green algal eyespot apparatuses have a high intramembrane
particle density in the plasma membrane and the outer chloroplast
envelope (Melkonian and Robenek, 1984) and that proteins
are associated with the carotenoid-rich eyespot globules
(Renninger et al., 2001, 2006). Thus, specific enrichment of
proteins intrinsic to this complex cell organelle can be expected
in the purified fraction. In this study, we intended to purify the eyespot
apparatus in its entire complexity along with the specialized
areas of the plasma membrane, chloroplast envelope, and thylakoid
system belonging to the functional eyespot apparatus. However,
complete separation of these specialized membrane areas
of the eyespot from the nonspecialized parts of these membrane
systems cannot be achieved by biochemical methods. Thus, we
cannot rule out that a small portion of membrane extensions is still
present in the final fraction. Additionally, only a few free membrane
patches not associated with the eyespot fragments were observed
in the electron microscopy analysis. Thereby, normal
constituents of such membranes and, to a lesser degree, also
from the stromal and cytosolic compartments could be present
among the proteins associated with this fraction.
Proteome Analysis of the Eyespot Apparatus
General Overview
To identify individual proteins of the enriched eyespot fraction by
MS/MS, proteins were separated by SDS-PAGE and the gel was
sliced into 54 pieces (see Supplemental Figure 1B online).
Proteins from half of each slice were in-gel digested with trypsin.
The generated peptide fragments were subjected to nano-LC-
ESI-MS/MS analyses using a linear ion trap mass spectrometer.
Table 1 summarizes the identified proteins (202 in total) along
with the number of different peptides that were found within a
given protein, their biological function (if known), and the presence
of predicted transmembrane domains (TMDs). Only proteins
are listed where at least two different peptides fulfilling the
criteria for the Xcorr, the probability score, and the dCN values
(see Methods) could be identified by peptide MS/MS using
SEQUEST-based Bioworks software (version 3.2) along with
Chlamydomonas databases. From the 202 proteins identified with
high confidence, 72 proteins were identified by five or more
different peptides and 130 proteins by two to four different
peptides (Table 1). All different peptides (984 in total) from these
proteins are listed in Supplemental Table 1 online along with the
charges of the peptides, their Xcorr values, and the GRAVY index
of the proteins. The frequency distribution of the GRAVY index
(Figure 4) indicates enrichment of proteins with a more hydrophobic
character in the eyespot fraction. Similar GRAVY frequency
distributions have been reported for typical membrane
proteomes (e.g., the Arabidopsis plasma membrane and
subfractions of the thylakoid membrane; Friso et al., 2004;
Marmagne et al., 2004). Also, the TMD predictions for the 202
proteins corroborate enrichment of proteins with a hydrophobic
character. Thirty-nine proteins (19.3%) were predicted to contain
TMDs by all three used programs (see Methods; Table 1), and for
another 80 proteins (39.6%), two programs predicted their
presence (i.e., these proteins have at least a partial hydrophobic
character). The enrichment of proteins with a moderate hydro-
Chlamydomonas Eyespot Proteome 11 of 23
Figure 4. The Majority of the Proteins from the Eyespot Proteome Have
a More Hydrophobic Character.
Frequency distribution of the GRAVY index of the proteins identified with
at least two peptides in the fraction enriched in eyespot apparatuses.
phobic and amphiphatic character correlates well with the ultrastructure
of the green algal eyespot apparatus, which is
dominated by hydrophobic structures.
All six currently known or predicted proteins from C. reinhardtii
related to the eyespot apparatus (for detailed discussion, see
section on known/predicted eyespot proteins and retinal-related
proteins) are among the identified proteins, indicating that the
presented proteome data might enclose a rather complete list.
Furthermore, with the exception of one of the retinal-based
photoreceptors (COP4; three different peptides), these proteins
were represented with 5 to 10 different peptides. As the number
of peptides identified in complex mixtures by ESI-MS/MS can
roughly correlate with the abundance and size of proteins
(Washburn et al., 2001), this further supports our conclusion
that eyespot proteins are indeed well enriched in the analyzed
fraction. However, we cannot rule out that some of the soluble
eyespot-related proteins might have been lost during the purification
procedure. As expected, a significant proportion of
proteins (at least 36%) also represent proteins of thylakoids
and chloroplast envelope membranes, which are part of the

12 of 23 The Plant Cell
Figure 5. Proteins with PAP-Fibrillin Domains Are Enriched in the Eyespot Fraction and Some Are up to Four Times Larger Than Fibrillins Associated
with Higher Plant Thylakoids and Plastoglobules.
(A) Polypeptides (N to C termini) identified in our MS analysis are represented schematically. PAP-fibrillin domains that were identified by National
Center for Biotechnology Information (NCBI) BLASTp conserved domain searches are indicated in black and their length is given below in amino acids
(aa). The reduced PAP-fibrillin domains observed in C_13870001 and C_2690006 are identical.
(B) Correlation between protein length (in amino acids) and the GRAVY index for those proteins containing PAP-fibrillin domains. Letters refer to the
gene model given in (A).
eyespot apparatus (Figures 1B and 1C). These include, for
example, many of the thylakoid membrane–associated proteins
of photosystems I and II along with its light-harvesting proteins
and the ATPase complex, proteins responsible for translocation
over the two chloroplast envelope membrane or plastidic chaperones.
By contrast, only a few cytosolic and stromal proteins
involved in primary carbon metabolism were identified. Notably,
the dominating ribulose-1,5-bisphosphate carboxylase/oxygenase
was not detected. Thirty proteins (14.9%) in the eyespot
fraction represented novel and conserved proteins of as yet
unknown function. Additionally, the list of proteins identified
by two or more peptides was enriched in proteins potentially
involved in signaling (9.9%), proteins possessing plastid lipidassociated
protein (PAP)-fibrillin domains (4%), and in proteins
involved in pigment biosynthesis (4.5%) and lipid metabolism
(3.5%). The latter two include also rather specialized enzymes of
such pathways. These will be discussed in detail later. Only one
protein of the cytoskeleton, a-tubulin, was identified with two
peptides. Major contaminants appear to come from cytosolic ribosomes
and DNA-related proteins (8.4%), mitochondria (6.9%),
and the Golgi/endoplasmic reticulum/vesicle trafficking machinery
(3%). Proteins belonging to the latter two groups probably
arise from free membrane patches that were not associated with
the eyespot fragments, as mentioned before. These values are in
the contamination range reported for proteome analysis of other
cell organelles rather subtle in isolation, for example, the vegetative
vacuole of Arabidopsis (Carter et al., 2004) or the basal
bodies of C. reinhardtii (Keller et al., 2005).
An additional 381 proteins were identified by only one peptide
(see Supplemental Table 2 online). This group of proteins was not
subjected to an in-depth analysis. It contains likely contaminants
and small and/or very low abundance proteins possibly related
with the eyespot apparatus.
Known/Predicted Eyespot Proteins
and Retinal-Related Proteins
As already mentioned, our data set contains all currently known
or predicted proteins related to the eyespot apparatus. Besides
the two retinal-based photoreceptors, COP3 and COP4 (eight
and three different peptides, respectively), that are involved in
phototactic and photophobic responses (Nagel et al., 2002,
2003; Sineshchekov et al., 2002; Suzuki et al., 2003), both
splicing variants of the abundant retinal binding protein COP
(COP1 and COP2; Deininger et al., 1995; Fuhrmann et al., 2003)
were identified with five peptides. Both proteins seem not to be
involved in behavioral responses, and their function is still unclear
(Fuhrmann et al., 2001). Localization of COP1/2 and COP3
to the eyespot apparatus was previously demonstrated by
immunofluorescence and/or green fluorescent protein tagging

Figure 6. CK1 Is Enriched in Eyespot and Flagella Fractions.
(A) Proteins from a crude extract (CE; 8 mg per lane) and overexpressed
His-tagged CK1 lacking the N terminus (OX; 1.5 ng per lane) were
separated on SDS-PAGE along with a molecular mass standard, blotted,
and probed with antipeptide CK1 antibodies (anti-CK1) and preimmune
serum (PI), respectively. The position of CK1 is indicated with a black
arrow. Its determined molecular mass (;37 kD) differs only slightly from
its theoretical molecular mass (38.4 kD). In the case of the E. coli–
overexpressed CK1, its determined molecular mass (;26.5 kD; gray
arrows) also differs only minimally from its theoretical molecular mass
(25.9 kD).
(B) Proteins from a crude extract (CE; 4, 8, and 16 mg per lane), an
eyespot (ES; 4 mg per lane), and a flagella fraction (F; 4 mg per lane) were
separated on SDS-PAGE along with a molecular mass standard and
immunoblotted with antipeptide CK1 antibodies. The position of CK1 is
indicated with a black arrow.
in C. reinhardtii (Deininger et al., 1995; Fuhrmann et al., 1999;
Suzuki et al., 2003). The theoretical postulated additional photoreceptors
that show sequence homology to conserved domains
of COP1-4 (Kateriya et al., 2004) were, however, not found
in our data set. EYE2 and MIN1, two proteins important for
eyespot formation and size control (Lamb et al., 1999; Roberts
et al., 2001; Dieckmann, 2003), were identified with 7 and 10
peptides, respectively. EYE2 has been shown by protein gel blot
analysis to be enriched in a fraction of intact eyespot apparatuses
from S. similis, but was not detectable in purified eyespot
globules from this green alga (Dieckmann, 2003; Renninger et al.,
2006). Recently, insertions alleles of two other mutants (eye3 and
mlt1) that cause defects in eyespot development have been
identified (Lamb et al., 1999; Dieckmann, 2003). It will be interesting
to check whether these gene products will be among the
identified proteins once their sequences will be known and
released to public.
Beside proteins involved in the general biosynthesis pathway
of carotenoids in C. reinhardtii (e.g., 1-deoxy-D-xylulose-5phosphate
synthase and phytoene desaturase; Grossman et al.,
2004), we also identified proteins that are possibly important for
retinal biosynthesis in the eyespot proteome. One protein
(C_80229) has similarities to cyanobacterial lingostilbene-a,
b-dioxygenases and b-carotene-15,159-monooxygenases. Whereas
the latter enzymes are known to produce retinal from b-carotene,
it has been recently shown that an enzyme from Synechocystis
sp PCC 6803 annotated as lingostilbene-a,b-dioxygenase forms
retinal from diverse apo-carotenoids (Ruch et al., 2005). The
Chlamydomonas Eyespot Proteome 13 of 23
protein identified in the eyespot fraction has an almost identical
molecular mass (54.1 kD) as the Synechocystis protein (54.3 kD)
and appears to be quite abundant (12 different peptides). Another
protein (C_2440006) has similarities to a retinol dehydrogenase
and other members of the oxidoreductase short-chain
dehydrogenase/reductase family. In the visual cycle of vertebrates,
these enzymes can catalyze in principle the retinol or
retinal synthesis step (e.g., Palczewski and Saari, 1997). The
detection of these two enzymes in the eyespot fraction indicates
that at least part of retinal biosynthesis might take place directly
in the region of the eyespot apparatus (i.e., in close vicinity to the
retinal-based photoreceptors).
A third protein (C_970031) with similarities to the SOUL/HBP
family of proteins was detected with 14 peptides in the eyespot
proteome. In vertebrates, the SOUL protein is specifically expressed
in the retina and pineal gland. However, its physiological
role in the eye is not known (Zylka and Reppert, 1999; Sato et al.,
2004). Members of the SOUL/HBP family have also been identified
in the genomes of Arabidopsis, rice (Oryza sativa), and
other higher plants, and a SOUL domain protein is present in
the plasma membrane proteome of Arabidopsis (Marmagne
et al., 2004). The protein identified in the eyespot fraction has
highest similarities to the SOUL-heme binding-like proteins from
Figure 7. Silencing of CK1 by RNAi Down to Levels Below 25% in
Comparison with the Wild Type.
(A) The RNAi construct used for silencing of CK1 is demonstrated. The
numbers represent the involved exons. Dark gray regions are introns.
The light gray area represents the 59 untranslated region. The potential
promoter region (prom.) comprises 780 bp in front of the ck1 gene.
(B) Different amounts of proteins from a crude extract (100, 50, and 25 mg
per lanes) of wild-type cells were separated on SDS-PAGE (large-scale
size) and used for protein gel blot analysis with the antipeptide CK1
antibodies along with proteins of crude extracts (100 mg per lane) from
different CK1-silenced strains (CK1-sil).

14 of 23 The Plant Cell
Figure 8. Characterization of CK1-Silenced C. reinhardtii Strains by
Light Microscopy.
(A) to (H) Differential interference contrast and phase contrast images of
CK1-sil 2 strain grown in TAP ([A] to [D]) or minimal medium ([E] to [H]).
Cells tend to form aggregates (palmelloids) enclosed by the mother cell
wall most likely due to defects in hatching ([A] and [E]). This tendency
increases with the level of CK1 silencing. For quantitative analysis, see
Table 2. Single cells also exhibit variation in flagella length. Cells with
normal ([B], [G], and [H]; for quantitative analysis, see Table 3), intermediate
(C), and flagella stumps ([D] and [F]).
(I) CK1-sil 42 strain grown in TAP medium.
(J) CK1-sil 51 strain grown in TAP medium.
Black arrows, flagella/flagella stumps; white arrows, eyespot; arrowheads,
mother cell walls. Bars ¼ 10 mm.
Arabidopsis and rice. However, functional information is not
available yet in plants.
Proteins with PAP-Fibrillin Domains Are Enriched in the
Eyespot Apparatus Fraction
None of the structural proteins preventing coalescence of the
carotenoid-rich eyespot globules in the highly ordered eyespot
globule plate are currently known. Potential candidates for such
functions may belong to the fibrillin family. Fibrillin, also termed
PAP, is a major protein of carotenoid-rich fibrils and plastoglobules
in chromoplasts and chloroplasts and plays an important
role in carotenoid sequestration (Deruère et al., 1994; Pozueta-
Romero et al., 1997). Proteins belonging to the fibrillin family are
characterized by the PAP-fibrillin domain (gnljCDDj16052; pfam
04,755) and constitute a conserved group of proteins present in
most plastid types. They are mainly localized at the interface
between lipids and the stroma (Rey et al., 2000). Their molecular
masses range typically between ;25 and ;45 kD. Thus, the
sizes of fibrillins associated with thylakoids/plastoglobules in
Arabidopsis varies between 234 and 409 amino acids, and their
conserved PAP-fibrillin domain sizes range from 151 to 217
amino acids (Friso et al., 2004). In the fraction enriched in eyespot
apparatuses, eight proteins with PAP-fibrillin domains were
identified (Table 1). Five of these proteins fall in this size range,
whereas three are significantly larger (800 to 1788 amino acids;
Figure 5A). In addition, two proteins (C_2690006 and C_13870001)
that have an unusual small PAP-fibrillin domain (27 amino acids)
were found. Notably, all PAP-fibrillin domain containing proteins
in the eyespot fraction have a hydrophobic character, but the two
largest are more hydrophobic than the others (Figure 5B).
One function of members of the fibrillin family in higher plants is
stabilization of carotenoid fibrils, plastoglobules, and thylakoids
(Deruère et al., 1994; Gillet et al., 1998; Eymery and Rey, 1999;
Kessler et al., 1999; Rey et al., 2000). Most notably, overexpression
of fibrillin in higher plants resulted in organization of
plastoglobules in clusters, whose degree appears to be correlated
with the abundance of this protein. Also, fibrillin was
suggested to prevent coalescence of plastoglobuli and to mediate
interactions between carotenoid-rich fibrils (Deruère et al.,
1994; Rey et al., 2000). Therefore, the presence of several
proteins with PAP-fibrillin domains in the eyespot proteome is
of special interest with regard to its structure. In the functional
eyespot apparatus, the carotenoid-rich globules exhibit a close
hexagonal packing and have an intimate contact to thylakoids
and the chloroplast envelope. Thus, we hypothesize that some of
the proteins with PAP-fibrillin domains might have functions in
globule stabilization and may also be involved in interactions
necessary to form the highly ordered eyespot globule layers. The
presence of a specialized interface between the eyespot globules
and toward the stroma is supported by thin section and
freeze fracture electron microscopy, which demonstrated regularly
arranged particles at the eyespot globule surface in different
green algal species (Walne and Arnott, 1967; Renninger et al.,
2001). In addition, biochemical evidence for the involvement of a
protein scaffold in globule stabilization and globule–globule as
well as globule–membrane interactions has been provided by the
use of proteases (Renninger et al., 2001, 2006). However, not all
proteins possessing this domain might have these functions.
One of the PAP-fibrillin domain proteins identified in the eyespot
proteome (C_2460003) has similarities to higher plant proteins
annotated in the databases as harpin-interacting proteins.
Harpins are proteins produced by bacterial plant pathogens,
which elicit the complex natural defense mechanisms in plants
(Wei et al., 1992) and also possess a PAP-fibrillin domain.
Table 2. Silencing of CK1 Affects Hatching
Growth
Medium Strain
Single
Cells
Percentage of
Aggregates
of Two Cellsa Aggregates of More
Than Two Cells
Minimal medium b CK1-sil 2 54.3 34.2 11.5
CK1-sil 42 9.0 21.0 70.0
CK1-sil 51 2.7 9.3 88.0
TAP CK1-sil2 19.2 15.8 65.0
CK1-sil 42 12.8 20.2 67.0
CK1-sil 51 2.6 9.0 88.4
a n ¼ 292 to 500 analyzed cells/cell aggregates.
b Analyses were done 24 h after transfer of cells from TAP to minimal
medium.

Table 3. Silencing of CK1 Affects Flagella Formation
Growth Medium Strain
Minimal medium
TAP a
CK1-sil2 CK1-sil42 CK1-sil51 CK1-sil2 CK1-sil42 CK1-sil51 Percentage of Single Cells
with Normal Flagella
80.2
37.0
16.7
31.7
7.5
0.6
a Cultures were treated with autolysin for 15 to 20 min to release cells
from palmelloids before fixation.
Beside the PAP-fibrillin domain-containing proteins, two further
potential candidates for mediating membrane–membrane interactions
came up in the eyespot proteome. The first is encoded by
gene model C_840016 and has similarities to a cell adhesion
protein from the colonial green alga Volvox carteri. In addition, this
protein contains two weak fascilin I domains. These domains are
present in cell adhesion molecules of vertebrates, invertebrates,
plants, and bacteria. The second protein (C_190173) contains a
region with similarities to an algal MORN repeat protein. The
MORN repeat motif functions in attaching proteins to membranes
and forming junctional complexes between membranes
(Takeshima et al., 2000; Shimada et al., 2004). In the eyespot
apparatus, proteins mediating membrane–membrane interactions
might be involved in, for example, maintaining the close contact
between the plasma membrane and the chloroplast envelope.
Ca 21 Binding Proteins, Kinases, and Phosphatases Are
Present in the Eyespot Apparatus
The eyespot apparatus acquires light information via photoreceptors
and forwards it through signaling pathways to the flagella. In
these signaling cascades, Ca 2þ is intricately involved (Witman,
1993; Pazour et al., 1995; Sineshchekov and Govorunova, 1999).
Excitation of the photoreceptors in the eyespot apparatus initiates
fast inward directed complex photoreceptor currents in the
eyespot region, which are mainly carried by Ca 2þ (Harz and
Hegemann, 1991; Holland et al., 1996). COP3 and COP4 are
directly light-gated channels allowing an extreme fast depolarization
at high light intensities (Nagel et al., 2002, 2003). At low light
intensities, however, delay of the photoreceptor currents by
several milliseconds suggests involvement of a signal amplification
system indirectly activating ion channel activity in the eyespot
apparatus (Braun and Hegemann, 1999; Sineshchekov and
Govorunova, 2001). As COP4 is mainly permeable to Ca 2þ
(Nagel et al., 2003), an early increase in the free concentration
of Ca 2þ in the narrow space between the plasma membrane and
chloroplast envelope in the region of the eyespot apparatus can
be expected to be involved in signaling. In accordance with the
major role of Ca 2þ fluxes in both photoresponses, we identified
five proteins with potential roles in Ca 2þ signaling in the eyespot
Chlamydomonas Eyespot Proteome 15 of 23
proteome. One protein (C_1010018, eight different peptides) is
annotated as a calcium sensing receptor of C. reinhardtii, and the
other four proteins have Ca 2þ binding domains belonging to the
EF-hand superfamily (two to five different peptides). For these
proteins, similarities in BLAST searches are restricted to the Ca 2þ
binding domains. The hydrophobic character of all five proteins
favors their local restriction to membranes in the region of the
eyespot apparatus. Thus, these proteins are potential candidates
for spatially restricted Ca 2þ signaling processes likely to occur
upon stimulation of the photoreceptors. In accordance with this
suggestion, these proteins are absent from the flagella, in which
Ca 2þ also plays a major signaling role (Witman, 1993; Pazour
et al., 2005).
Changes in the free concentration of Ca 2þ from 10 ÿ8 to 10 ÿ7 M
have been shown to strongly affect rapid protein phosphorylation
and dephosphorylation in isolated green algal eyespot apparatuses
(Linden and Kreimer, 1995). Based on homology searches
and domain analyses, four kinases and two phosphatases were
identified in the eyespot proteome (Table 1), underlining the
potential importance of protein phosphorylation/dephosphorylation
in signaling processes in the eyespot apparatus. Two
proteins (C_230061 and C_110160) are defined by their AarF
domain as members of a group of unusual protein kinases. The
other two gene models encode known protein kinases: the cyclic
nucleotide-dependent protein kinase II (C_60149) and CK1
(C_70149). In addition, the blue light photoreceptor phototropin
was identified in the eyespot proteome. As a member of the
phototropin family, this C. reinhardtii protein contains a Ser-Thr
kinase domain (Huang et al., 2002). Interestingly, these three
proteins are also localized in the flagella (Huang et al., 2004;
Pazour et al., 2005), pointing to their importance for motility and
Figure 9. The Level of CK1 Is Significantly Reduced in Flagella of CK1sil
2 and Eyespot Fractions of CK1-sil 2 and CK1-sil 51.
(A) Proteins from a crude extract of wild-type cells (CE; 4 and 8 mg per
lane) and the eyespot (ES; 8 mg per lane) and flagella fractions (F; 8 mg
per lane) of CK1-sil 2 were separated on SDS-PAGE along with a
molecular mass standard and immunoblotted with antipeptide CK1
antibodies.
(B) Proteins from a crude extract of wild-type cells (CE; 4 and 8 mg per
lane) and the eyespot fraction (ES; 8 mg per lane) of CK1-sil 51 were
separated on SDS-PAGE along with a molecular mass standard and
immunoblotted with antipeptide CK1 antibodies.
Black arrows indicate the position of CK1. Exposure time was lengthened
in comparison with Figure 6B to allow detection of low amounts of
CK1 in the subcellular fractions of the CK1-silenced strains.

16 of 23 The Plant Cell
Table 4. Wild-Type and CK1-sil 2 Cells Show Diurnal
Phototactic Behavior
Time Strain
LD 6 a Wild type 290.0
LD 8 a Wild type 265.0
LD 18 b Wild type 17.5
LD 20 b Wild type 22.5
LD 6 a CK1-sil 2 109.0
LD 8 a CK1-sil 2 96.0
LD 18 b CK1-sil 2 26.0
LD 20 b CK1-sil 2 24.5
Extinction (mV) Reflecting
the Amount of Cells That
Swim to the Light
a LD 6 and LD 8: 6 or 8 h after light was switched on in a 12-h-light/12-hdark
cycle representing the middle of the day;
b LD 18 and LD 20: 6 or 8 h after light was switched off in a 12-h-light/12h-dark
cycle representing the middle of the night.
possibly also tactic responses. CK1 experimental evidence for
this assumption will be presented later.
Based on the high number of different peptides, it can be
assumed that the two Ser-Thr phosphatases encoded by the
gene models C_760036 (37 different peptides) and C_760032 (22
different peptides) are rather dominating proteins in the eyespot
fraction. Both are members of the PP2C family of phosphatases.
Different PP2Cs have been found as regulators of signal transduction
pathways and development in plants (Schweighofer
et al., 2004). Their apparent dominance may point to the need of
rapid downregulation of signaling pathways initiated by protein
kinases in the eyespot apparatus. This assumption would be in
accordance with the recurrent theme in higher plants that PP2Cs
regulate signaling negatively (Schweighofer et al., 2004). As with
the Ca 2þ binding proteins, their moderate hydrophobic character
might allow special restriction to the region of the eyespot
apparatus. These phosphatases are not present in the flagella,
where massive protein phosphorylation and dephosphorylation
is crucial for signaling and motility control (Porter and Sale, 2000;
Pazour et al., 2005).
Functional characterization of these putative signaling elements
may aid future research in unraveling the signaling mechanisms
starting from the eyespot apparatus. However, it should
be considered that some soluble and low abundance proteins
might be missing in the list of putative signaling proteins present
in this complex cell organelle.
Possible Chemotaxis-Related Proteins in the
Eyespot Apparatus
C. reinhardtii exhibits chemotactic responses to various substances.
Its sensitivity to chemical stimuli is tightly related to its
life cycle and is controlled by blue light. Phototropin has been
shown to control multiple steps in the sexual life cycle of
C. reinhardtii and to play a crucial role in mediating changes
in chemotaxis during the initial phase of the sexual life cycle
(Huang and Beck, 2003; Ermilova et al., 2004; Govorunova and
Sineshchekov, 2005). As already mentioned, it is present in the
eyespot proteome. In addition, chemical stimuli interfere with the
inward photoreceptor currents, the earliest detectable events in
the signal transduction chain of the photoresponses, pointing to
a possible integration of photosensory and chemosensory signaling
pathways at their initial steps (Govorunova and Sineshchekov,
2003, 2005). In this context, the detection of a protein with
similarities to a cyanobacterial methyl-accepting chemotaxis
protein (MCP) in the eyespot proteome is of special interest.
Figure 10. Circadian Phototaxis Is Significantly Disturbed in Its Period in
the CK1-sil 2 Strain.
(A) Circadian phototaxis of wild-type strain SAG 73.72 was measured
using the automated phototaxis-measuring unit developed by
Mergenhagen (1984). ‘‘E’’ represents the extinction in millivolts. Time
(days) indicates how long cells were exposed to constant darkness. The
free-running period of the wild type (24.5 h) is indicated.
(B) Phototaxis of a control strain that was transformed with the aphVIIIcontaining
vector pSI103 (period: 24.6 h) and of the strain CK1-sil 2
(period: 23 h over the first 4 to 5 d, then tendency to arrhythmicity).

MCPs are the receptor proteins in bacterial chemotaxis (Szurmant
and Ordal, 2004). The protein, encoded by gene model
C_1250029, is completely covered by ESTs and exhibits a
weak similarity to a MCP of Nostoc. It was identified by five
different peptides in our analysis. Its molecular mass (18.8 kD) is
slightly larger than that of the Nostoc protein (11.7 kD). Of the 107
aligned amino acids, 44.9% are identical and 21.5% are in
addition functionally conserved between both proteins. Notably,
two proteins (C_290078 and C_390049) with similarities to
cyanobacterial methyltransferases (three and four different peptides,
respectively) also were identified along with the MCP-like
protein in the eyespot fraction. In bacterial chemotaxis, methylation
and demethylation of MCPs is important for sensory
adaptation and provide a memory mechanism in chemotaxis
(Webre et al., 2003; Szurmant and Ordal, 2004). However, the
involvement of these proteins in chemosensory signaling of
C. reinhardtii remains to be demonstrated experimentally.
Proteins of the Eyespot Apparatus That Might Be
Involved in Circadian Regulation
Both tactic movements (phototaxis and chemotaxis) in
C. reinhardtii are controlled by the circadian clock (Bruce,
1970; Mergenhagen, 1984; Byrne et al., 1992). Therefore, it
seems likely that the photoreceptor(s) relevant for circadian
control and clock-related signaling components are located in or
close to the eyespot. When searching the eyespot proteome for
proteins that might be involved in the circadian input/signaling
pathways, two candidates came up. One of them is the already
mentioned blue light photoreceptor phototropin. The presence
of blue light receptors in or close to the eyespot apparatus was
not considered until now. Additionally, phototropin has not been
characterized with regard to a possible circadian function. However,
it is a potential candidate for a circadian photoreceptor in
C. reinhardtii since physiological studies have shown that blue
light (besides red light) can reset the phase of circadian phototaxis
(Johnson et al., 1991; Kondo et al., 1991). Of course, this
has to be experimentally analyzed in the future. Notably, phototropin
is additionally located in the flagella (Huang et al., 2004;
Pazour et al., 2005) as pointed out earlier.
The other protein found in the eyespot proteome that could
represent a circadian-related signaling component is the abovementioned
CK1. Similar to phototropin, it is additionally located
in the flagella (Yang and Sale, 2000; Pazour et al., 2005). Casein
kinases belong to the Ser-Thr kinases. In Drosophila and mammals,
CK1 is involved in the phosphorylation of PERIOD, a key
oscillatory component thus gating the circadian feedback loop
(Panda et al., 2002; Reppert and Weaver, 2002). In Drosophila,
mutations of CK1 (known as DBT) either shorten or lengthen the
period of circadian rhythms (Preuss et al., 2004). CK1 is highly
conserved in C. reinhardtii as well as other circadian relevant
Ser-Thr kinases (CK2 and SHAGGY) and phosphatases (PP1 and
PP2A; Mittag et al., 2005). In the eyespot proteome, these other
kinases and phosphatases were not found. However, two Ser-
Thr phosphatases of type PP2C were present in this proteome,
while PP1 and PP2A appear in the flagella proteome (Pazour
et al., 2005).
Chlamydomonas Eyespot Proteome 17 of 23
CK1 Influences Several Processes, Including Hatching,
Flagella Formation, and Circadian Phototaxis
CK1 represents a bona fide candidate as member of a circadian
signaling cascade transducing light information that is taken up
by one of the photoreceptors in the eyespot to the flagella, thus
mediating circadian phototaxis and chemotaxis. Therefore, we
decided to select at first CK1 among the eyespot proteins for
further functional analysis.
Presence of CK1 in the Eyespot and the Flagella
In the eyespot proteome, CK1 was only identified via two peptides.
To verify enrichment of this kinase in the eyespot, we conducted
protein gel blot analyses with antipeptide CK1 antibodies. Selectivity
of the antibodies toward CK1 (theoretical molecular mass of
38.4 kD) was analyzed by comparison with preimmmune serum
and by loading a low amount (1.5 ng) of overexpressed His-tagged
CK1 lacking the N terminus (theoretical molecular mass 25.9 kD).
While the anti-CK1 peptide antibodies detected a protein of;37 kD
and the purified His-tagged CK1 fragment (determined molecular
mass of ;26.5 kD), these proteins were not recognized by the
preimmune serum (Figure 6A). We also examined the presence of
CK1 in a crude extract, an eyespot, and a flagella fraction. CK1
was clearly identified in the eyespot and the flagella fractions as
well as in the crude extract (Figure 6B). Furthermore, CK1 is
significantly enriched in the eyespot fraction and in the flagella in
comparison with a crude extract. Presence of CK1 in the eyespot
was verified in five independent preparations.
Silencing of CK1 Causes Period Shortening of the
Circadian Phototaxis Rhythm and a Tendency to
Arrhythmicity after 4 to 5 d
To obtain functional information about the role of CK1 in
C. reinhardtii, it was silenced by RNA interference (RNAi).
For silencing of CK1, an RNAi construct was created following
the strategy developed for C. reinhardtii by Fuhrmann et al.
(2001). Thereby, the potential endogenous promoter region of
ck1 (780 bp upstream of the gene) along with the 59 region
containing its 59 untranslated region and the first four exons,
including three introns, were fused to a cDNA fragment covering
exons 1 to 4 in opposite direction (39 / 59; Figure 7A). As selection
marker, the aphVIII gene encoding resistance to paromomycin
(Sizova et al., 2001) was used. Transformed strains
growing on paromomycin were used for further analysis. Cells
were grown up to a cell density of ;4 to53 10 6 cells/mL, and
crude extracts were prepared. For comparison, a crude extract
from nontransformed wild-type cells was used. CK1 silencing
was checked in protein gel blot analysis with the antipeptide CK1
antibodies (Figure 7B). Different amounts of proteins from the
wild type (100, 50, and 25 mg per lane) were separated on SDS-
PAGE and quantitatively compared with proteins from transformed
strains (100 mg per lane) after immunoblotting with the
CK1 antibody. Equal loading was checked by Ponceau staining.
While some strains showed only very little silencing of CK1 (CK1sil6,
CK1-sil48, and CK1-sil50), others were significantly silenced
down. Thus, CK1-sil 2 was silenced to a level between 25 and

18 of 23 The Plant Cell
40%; CK1-sil 42 and CK1-sil 51 were silenced even below 25% in
comparison with the wild-type level of CK1.
The three well-silenced strains (CK1-sil 2, CK1-sil 42, and
CK1-sil51) were maintained and further analyzed by differential
interference contrast and phase contrast microscopy. These
analyses revealed that CK1 silencing causes multiple defects
that depend on the degree of silencing. In all three stains,
hatching (Figure 8, Table 2) and flagella formation (Figure 8, Table
3) were disturbed. The defects were more pronounced in Trisacetate-phosphate
(TAP) medium (used for usual culturing) than
in minimal medium (used for phototactic measurements). In the
CK1-sil 2 strain (silencing between 25 and 40%), 19.2% of cells
(TAP medium) and 54.3% of cells (minimal medium) occurred as
single cells, while the others occurred as pallmeloids (aggregates
of two or more cells within the mother cell wall). This phenotype is
indicative for defects in hatching. In addition, flagella formation
was affected. Only 31.7% (TAP) and 80.2% (minimal medium) of
the cells had normal flagella. Others had either medium sized
flagella, flagella stumps, or a bald phenotype. In the CK1 RNAi
strains sil 42 and sil 51 that are silenced below 25%, the disturbances
in hatching and flagella formation were even more
severe. Thus,

105 min, 48C), the orange-red bands at the interface of GSS and 20.5%
sucrose were collected, brought to 25% (w/v) sucrose, and further
purified by flotation centrifugation (100,000g, 60min,48C) on discontinuous
sucrose gradients (22 mL sample, 7 mL 15% [w/w] sucrose, 7 mL
4% [w/w] sucrose, and 2 mL GSS). The deep orange bands at the 4 and
15% sucrose interfaces were collected, brought again to 25% (w/v)
sucrose, and further purified by repeating the above-mentioned flotation
gradient centrifugation step. For concentration, the fraction was brought
to 25% (w/v) sucrose, layered on 16 mL 42% (w/w) sucrose, and overlaid
by 2 mL GSS and centrifuged again (100,000g, 30 min, 48C). The
concentrated eyespot fraction was collected from the top of the gradient,
directly extracted with chloroform:methanol:water (4:8:3), and dissolved
in 23 SDS sample buffer (Kreimer et al., 1991).
Thylakoids were isolated according to Chua and Bennoun (1975) with
the following modifications. Cell concentration and disruption was done
as described above. The cell homogenate was diluted 1:1 with 5 mM
HEPES, pH 7.5, and centrifuged (2000g,10min,48C), and the pellet was
resuspended in 5 mM HEPES, pH 7.5. After centrifugation (46,000g,
12 min, 48C), the pellet was resuspended in 1.8 M sucrose buffered with
5 mM HEPES, pH 7.5, and homogenized in a 25 mL potter with 20 strokes.
Thylakoids were separated by discontinuous sucrose gradients buffered
with 5 mM HEPES, pH 7.5, consisting of 13 mL sample, 7 mL 1.3 M
sucrose, and 18 mL 0.5 M sucrose. After centrifugation (100,000g, 60 min,
48C), thylakoids from the 1.3 M sucrose region were collected, diluted 1:4
with 5 mM HEPES, pH 7.5, and concentrated by centrifugation (46,000g,
12 min, 48C). The pellets were stored at ÿ808C until further use.
Electrophoretic Methods
For SDS-PAGE analysis, a modified high Tris system was used. Proteins
were separated either with a medium sized or by large-scale gel systems.
Lipid removal, protein precipitation, and SDS-PAGE were conducted as
described (Kreimer et al., 1991; Calenberg et al., 1998). Gels were either
stained with standard Coomassie Brilliant Blue G 250, Bio-Safe Coomassie
(Bio-Rad), or silver (Rabilloud et al., 1988; Wagner et al., 2004).
Images of gels and blots were either scanned or taken with a Coolpix 990
(Nikon) and processed with Photoshop (Adobe Systems).
MS Analysis
In-Gel Digestion
The gel was dissected into 54 bands. Gel slices were washed for 10 min
with 10 mM NH 4HCO 3 and then 10 min with 5 mM NH 4HCO 3/50% (v/v)
acetronitril. These steps were repeated two times. Gel slices were
vacuum dried and stored at ÿ808C. Trypsin (20 mg; Promega) was
resuspended in 40 mL 50 mM acetic acid and diluted with 50 mM
NH 4HCO 3 to a final concentration of 60 ng/mL. Each gel slice was cut into
two pieces. The 30 mL trypsin solution was added to one of these gel
pieces and incubated for 20 min on ice. Residual solution was removed,
and gel slices were covered with 50 mM NH 4HCO 3 and incubated at 378C
overnight. The supernatant was collected, and gel slices were washed
two times for 1 h with 50 mL acetonitril:water (3:2). All supernatants were
combined and vacuum dried.
Nano-LC-ESI-MS/MS
The pellet was resuspended in 5 mL 5% (v/v) acetonitril/0.1% (v/v) formic
acid and subjected to nano-LC-ESI-MS/MS using an UltiMate 3000 nano
HPLC (Dionex) with a flow rate of 300 nL/min coupled online with a linear
ion trap ESI mass spectrometer (Finnigian LTQ ; Thermo Electron). A
gradient was used to elute peptides from the reverse phase C18 column
(LC Packings). The successive steps of the gradient were as follows: 5 min
4% A/96% B (v/v); within 30 min gradually to 50% A/50% B (v/v); within
1 min gradually to 90% A/10% B (v/v); 5 min 90% A/10% B (v/v); within
1 min gradually to 4% A/96% B (v/v); 18 min 4% A/96% B (v/v); whereby A
consists of 0.1% (v/v) formic acid in water and B consists of 0.1% (v/v)
formic acid in acetonitril. The mass spectrometer was cycling between
one full MS and MS/MS scans of the four most abundant ions. After each
cycle, these peptide masses were excluded from analysis for 3 min.
Data Analysis
Data analysis was done with Bioworks software (version 3.2; Thermo
Electron) including the SEQUEST algorithm (Link et al., 1999). Searches
were done for tryptic peptides, allowing two missed cleavages. The
software parameters were set to detect a modification of 16 D on Met
representing its oxidized form. Scores for the cross-correlation factor
X corr (Eng et al., 1994) were set to the following limits: X corr > 1.5 if the
charge of the peptide is 1; X corr > 2 if the charge of the peptide is 2; X corr >
2.5 if the charge of the peptide is 3. Only peptides that fulfilled the Xcorr
limits and also had a peptide P value # 0.01 and a dCN $ 0.081 were
included in the tables. P was only recently introduced with the new
Bioworks (version 3.2) software and represents the statistical likelihood of
finding an equally good peptide match by chance. By default, peptide
probabilities are reported as probabilities normalized to 1, and a lower
probability value represents a better match (Bioworks, version 3.2).
Application of a P value screening, in addition to the Xcorr and dCN
settings, strengthens the quality of positive hits.
Data were searched against the following C. reinhardtii databases: final
model database from the Joint Genome Institute (version 2; http://
genome.jgi-psf.org/chlre2/chlre2.home.html], mitochondrial database
available from the NCBI (NC001638; gi:11467088), and the chloroplast
database (www.chlamy.org/chloro/default.html). Data from all runs were
combined and further evaluated using a program developed in-house.
The peptide sequences of the gene models were compared with the NCBI
protein database using BLAST (Altschul et al., 1997). For positive identification
of both protein and functional domain prediction, an internal
cutoff E-value of 1e-05 was used. A few exceptions were made in case of
specific functional implications (marked by an ‘‘h’’ in the tables). TMD
information was based on predictions by the programs TMHMM (Krogh
et al., 2001), TMpred (Hofmann and Stoffel, 1993), and TopPred (von
Heijne, 1992). The GRAVY index and number of amino acids were
determined with ProtParam (Gasteiger et al., 2005).
Silencing of CK1 via RNAi
Construction of the RNAi Vector
Chlamydomonas Eyespot Proteome 19 of 23
The potential promotor region of ck1 (780 bp in front of the gene) and the
first four exons and three introns of ck1 (gene model C_70149) were PCR
amplified using the GC-RICH PCR system kit (Roche Applied Science)
according to the manufacturer’s instructions along with genomic DNA from
C. reinhardtii and the following primers: OMM232 (sense direction:
59-AGGTATGCGTGCACAAAGTC-39) and OMM249 (antisense direction:
59-ATGAGCACCGTCTTGAGACTG-39). The genomic DNA was cloned into
the pCAP S vector from the PCR cloning kit (Roche Applied Science)
following the manufacturer’s instructions, and the resulting plasmid was
named pOV1. A piece of the ck1 cDNA was PCR amplified with SAWADY
Pwo DNA-polymerase (PEQLAB Biotechnology) using the primers
OMM228 (sense direction: 59-ATGGCGTTGGACATTCGGAT-39) and
OMM229 (antisense direction: 59-AACAGGTCGCGGAACATCTT-39) along
with a self-made cDNA library (Waltenberger et al., 2001) whose proteins
were removed by proteinase treatment. The cDNA was cloned into
pBluescript II KSþ, resulting in pGG1. The 258-bp cDNA fragment was
then fused in the opposite direction (39 / 59) by cutting pGG1 and pOV1

20 of 23 The Plant Cell
with ClaI andBamHI and inserting the fragment from pGG1 into pOV1,
resulting in pOV2. For selection in C. reinhardtii, the aphVIII gene (Sizova
et al., 2001) was introduced finally into the vector. Thereby, pSI103 containing
the aphVIII gene and pOV2 was cut with BamHI and ScaI. In the case
of pSI103, a partial digest with BamHI was performed. The 3437-bp
aphVIII-containing BamHI-ScaI fragment from pSI103 was then ligated into
the cut pOV2, resulting in pOV3. Vectors pGG1, pOV1, and pOV3 were
sequenced at Medigenomix to check the correctness of relevant sequences.
The ck1 cDNA and its genomic DNA were in accordance with
sequences from its EST assembly ACE 7.12.2.11 and gene model C_70149
together with its upstream genomic sequence, respectively. Cloning of the
aphVIII gene was also positively confirmed. All molecular biology procedures
were done according to Sambrook and Russell (2001).
Transformation of C. reinhardtii with the RNAi Vector
and Identification of CK1 by Protein Gel Blot Analysis
C. reinhardtii wild-type strain SAG 73.72 was grown in TAP medium under
a LD 12/12 cycle with a light intensity of 71 mmol m ÿ2 s ÿ1 at 248Cuptoa
cell density of 2 to 5 3 10 6 cells/mL for transformation. The 20 mg DNA of
pOV3 that was linearized with ScaI was used for transformation according
to Kindle (1990) with the following modification: Vortexing of the cells with
glass beads was performed for 15 s. Cells were then transferred in liquid
TAP medium in light overnight according to Davies et al. (1992) and plated
in TAP with 0.5% agar on paromomycin plates (selection medium)
following the protocol of Sizova et al. (2001). Cells were maintained on
TAP plates with paromomycin. For further culture in liquid medium, the
antibiotic was omitted.
Several colonies growing on the selection media were grown in TAP
medium along with wild-type cells, and crude extracts were prepared
according to Mittag (1996) with the two following modifications: (1) Cells
were vortexed for 5 3 1 min with glass beads; and (2) Complete
Proteinase Inhibitor Cocktail (Roche Applied Science) was added to the
extraction buffer according to the user’s manual. Protein gel blot analysis
was performed according to Zhao et al. (2004) with antipeptide CK1
antibodies. The antibodies were generated from Eurogentec. For immunization,
the following peptides coupled to key limpet hemocyanin
according to the Eurogentec protocol were used: H 2N-CLRFDDKPDY-
SYLRKM-CONH 2 and H 2N-HKKSGTVPRPAVPRVP-CONH 2. As secondary
antibody, monoclonal anti-rabbit immunoglobulin G clone RG-96
peroxidase conjugate (Sigma-Aldrich) was used, and the signals were
detected by chemoluminescence.
Overexpression of CK1 in Escherichia coli
For overexpression of CK1 in E. coli, a PCR fragment was amplified with
cDNA (Waltenberger et al., 2001) as template using the primers OMM231
(59-TATCGGCTTGGGCGCAAGATT-39) and OMM 272 (59-CCCAA-
TACCTGGTACCGTC-39). The PCR product was cut with KpnIandEcoRV,
and the resulting 977-bp fragment was inserted to pBluescript II KSþ that
was restricted with the same enzymes. This plasmid was named pGG2.
The ck1-encoding cDNA from pGG2 was then transferred to the pQE 30
overexpression vector (Qiagen). Therefore, pQE30 was digested with
BamH1 and SmaI and pGG2 with BamH1 and SseBI. Sequencing of the
resulting plasmid pGG3 showed a deletion of one nucleotide in the EcoRV
cloning site shifting the reading frame of CK1. Therefore, the ck1 cDNA
fragment was further cloned into pQE32 (Qiagen). For this purpose, pGG3
and pQE32 were digested with BamHI and HindIII, and the ck1-encoding
fragment was ligated into pQE32. The resulting plasmid, pGG4, encodes
for a 6x His-tagged protein including the C-terminal 203 amino acids of
CK1. Overexpression and purification of the His-tagged protein was
performed according to the instructions of the Qiagen manual. This
overexpressed protein has a theoretical molecular mass of 25.9 kD.
Automated Measurement of Circadian Phototaxis
(Photoaccumulation) with Wild-Type and RNAi Strains
The measurement was done with a self-made phototaxis machine developed
by Mergenhagen (1984). Preparation of cell culture for the assay,
phototaxis measurement, and data evaluation were done as described
(Mergenhagen, 1984). Briefly, cells were kept in minimal medium that
lacked acetate and contained ammonium as nitrogen source. Accumulation
of cells in an illuminated spot in the testing cuvette (30 mL flat Falcon
tube) was measured by a photocell. A light beam emitted by a light bulb
(Osram; 12 V, 500 mA) was focused by a two-lens condenser, passed
through the flask, and received by a photocell. The amount of light
transmitted through the Falcon flask depended on the number of cells in
the light path. The accumulation of the cells in the light path was read in
millivolts; no cells in the light path corresponded to 0 mV. Every 2 h, each
cuvette was illuminated for a period of 20 min using an electronically
stabilized voltage. The recording system was installed in a temperaturecontrolled
darkroom of 22.58C. For the assay, wild-type strain SAG 73.72,
a control strain that was transformed with the aphVIII-containing vector
pSI103, and the CK1-silenced strains were used.
Miscellaneous
Flagella were isolated basically according to King (1995). Fixation and
preparation of concentrated samples for electron microscopy were basically
done as described (Kreimer et al., 1991; Renninger et al., 2001). For
light microscopy, cells were fixed with 1% glutaraldehyde in TAP or
minimal medium. Protein content was measured accordingto Neuhoffet al.
(1979) or with the Bio-Rad protein assay with BSA as standard. Chlorophyll
and carotenoids were determined as described by Lichtenthaler (1987).
Accession Numbers
The gene and protein ID numbers listed in Table 1 are from Chlamydomonas
genome versions 2 and 3.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The Protein Pattern of the Fraction Enriched
in Eyespot Fragments Is Reproducible and Was Cut into 54 Slices for
Proteomic Analysis.
Supplemental Table 1. Identified Peptides of the Eyespot Apparatus
and Characterization of Their Corresponding Proteins.
Supplemental Table 2. Identified Peptides of the Eyespot Apparatus
with Only One Peptide per Protein.
ACKNOWLEDGMENTS
We thank Markus Fuhrmann for providing pSI103, Anne Mollwo and Eva-
Maria Schmidt for technical help, and Susan Hawat for initial help with MS
analysis. We also thank Frank Meißner for his help on bioinformatic
programming and Dieter Mergenhagen for the donation of the automated
phototaxis measuring unit to M.M. We appreciate the free delivery of information
by the U.S. Department of Energy genome project of C. reinhardtii.
This study was supported by grants from the Deutsche Forschungsgemeinschaft
to G.K. and M.M. and by a Deutscher Akademischer
Austauschdienst fellowship to O.V.
Received February 4, 2006; revised May 7, 2006; accepted June 1, 2006;
published June 23, 2006.

REFERENCES
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z.,
Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST:
A new generation of protein database search programs. Nucleic Acids
Res. 25, 3389–3402.
Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K.
(2005). Millisecond-timescale, genetically targeted optical control of
neural activity. Nat. Neurosci. 8, 1263–1268.
Braun, F.-J., and Hegemann, P. (1999). Two independent photoreceptor
currents in the spheroid alga Volvox carteri. Biophys. J. 76,
1668–1678.
Bruce, V.G. (1970). The biological clock in Chlamydomonas reinhardtii.
J. Protozool. 17, 328–334.
Byrne, T.E., Wells, M.R., and Johnson, C.H. (1992). Circadian rhythms
of chemotaxis to ammonium and methylammonium uptake in Chlamydomonas.
Plant Physiol. 98, 879–886.
Calenberg, M., Brohsonn, U., Zedlacher, M., and Kreimer, G. (1998).
Light- and Ca 2þ -modulated heterotrimeric GTPases in the eyespot
apparatus of a flagellate green alga. Plant Cell 10, 91–103.
Carter, C., Pan, S., Zouhar, J., Avila, E.L., Girke, T., and Raikhel, N.V.
(2004). The vegetative vacuole proteome of Arabidopsis thaliana
reveals predicted und unexpected proteins. Plant Cell 16, 3285–
3303.
Chua, N.-H., and Bennoun, P. (1975). Thylakoid membrane polypeptides
of Chlamydomonas reinhardtii: Wild-type and mutant strains
deficient in photosystem II reaction center. Proc. Natl. Acad. Sci. USA
72, 2175–2179.
Davies, J.P., Weeks, D.P., and Grossman, A.R. (1992). Expression
of the arylsulfatase gene from the b 2-tubulin promoter in Chlamydomonas
reinhardtii. Nucleic Acids Res. 20, 2959–2965.
Deininger, W., Kröger, P., Hegemann, U., Lottspeich, F., and
Hegemann, P. (1995). Chlamyrhodopsin represents a new type of
sensory photoreceptors. EMBO J. 14, 5849–5858.
Deruère, J., Römer, S., D’Harlingue, A., Backhaus, R.A., Kuntz, M.,
and Camara, B. (1994). Fibril assembly and carotenoid overaccumulation
in chromoplasts: A model for supramolecular lipoprotein structures.
Plant Cell 6, 119–133.
Dieckmann, C.L. (2003). Eyespot placement and assembly in the green
alga Chlamydomonas. Bioessays 25, 410–416.
Dunlap, J.C. (1999). Molecular bases for circadian clocks. Cell 96,
271–290.
Dunlap, J.C., and Loros, J.J. (2004). The Neurospora circadian system.
J. Biol. Rhythms 19, 414–424.
Eng, J., McCormack, A.L., and Yates, J.R. (1994). An approach to
correlate tandem mass spectral data of peptides with amino acid
sequences in a protein database. J. Am. Soc. Mass Spectrom. 5,
976–989.
Ermilova, E.V., Zalutskaya, Z.M., Huang, K., and Beck, C.B. (2004).
Phototropin plays a crucial role in controlling changes in chemotaxis
during the initial phase of the sexual life cycle in Chlamydomonas.
Planta 219, 420–427.
Eymery, F., and Rey, P. (1999). Immunocytolocalization of two chloroplastic
drought-induced stress proteins in well-watered or wilted
Solanum tuberosum L. plants. Plant Physiol. Biochem. 37, 305–312.
Foster, K.W., and Smyth, R.D. (1980). Light antennas in phototactic
alga. Microbiol. Rev. 44, 572–630.
Friso, G., Giacomelli, L., Ytterberg, A.J., Peltier, J.-B., Rudella, A.,
Sun, Q., and van Wijk, K.J. (2004). In-depth analysis of the thylakoid
membrane proteome of Arabidopsis thaliana chloroplasts: New proteins,
new functions, and a plastid proteome database. Plant Cell 16,
478–499.
Fuhrmann, M., Deininger, W., Kateriya, S., and Hegemann, P. (2003).
Chlamydomonas Eyespot Proteome 21 of 23
Rhodopsin-related proteins, cop1, cop2, and chop1, in Chlamydomonas
reinhardtii. In Photoreceptors and Light Signaling, A. Batschauer, ed
(Cambridge, UK: Royal Society of Chemistry), pp. 124–135.
Fuhrmann, M., Oertel, W., and Hegemann, P. (1999). A synthetic gene
coding for the green fluorescent protein (GFP) is a versatile reporter in
Chlamydomonas reinhardtii. Plant J. 19, 353–361.
Fuhrmann, M., Stahlberg, A., Govorunova, E., Rank, S., and Hegemann,
P. (2001). The abundant retinal protein of the Chlamydomonas eye
is not the photoreceptor for phototaxis and photophobic responses.
J. Cell Sci. 114, 3857–3863.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R.,
Appel, R.D., and Bairoch, A. (2005). Protein identification and
analysis tools on the ExPASy server. In The Proteomics Protocols
Handbook, J.M. Walker, ed (Totowa, NJ: Humana Press), pp.
571–607.
Gehring, W. (2004). Historical perspective on the development and
evolution of eyes and photoreceptors. Int. J. Dev. Biol. 48, 707–717.
Gillet, B., Beyly, A., Peltier, G., and Rey, P. (1998). Molecular characterization
of CDSP 34 a chloroplastic protein induced by water deficit
in Solanum tuberosum L. plants and regulation of CDSP 34 expression
by ABA and high illumination. Plant J. 16, 257–262.
Govorunova, E.G., Jung, K.-H., Sineshchekov, O.A., and Spudich,
J.L. (2004). Chlamydomonas sensory rhodopsins A and B: Cellular
content and role in photophobic responses. Biophys. J. 86, 2342–
2349.
Govorunova, E.G., and Sineshchekov, O.A. (2003). Integration of
photo- and chemosensory signalling pathways in Chlamydomonas.
Planta 216, 535–540.
Govorunova, E.G., and Sineshchekov, O.A. (2005). Chemotaxis in the
green flagellate alga Chlamydomonas. Biochemistry (Mosc.) 70,
717–725.
Grossman, A., Lohr, M., and Im, C.S. (2004). Chlamydomonas
reinhardtii in the landscape of pigments. Annu. Rev. Genet. 38,
119–173.
Harmer, S.L., Satchidananda, P., and Kay, S.A. (2001). Molecular
bases of circadian rhythms. Annu. Rev. Cell Dev. Biol. 17, 215–253.
Harris, E.H. (1989). The Chlamydomonas Sourcebook. (San Diego, CA:
Academic Press).
Hartshorne, J.N. (1953). The function of the eyespot in Chlamydomonas.
New Phytol. 52, 292–297.
Harz, H., and Hegemann, P. (1991). Rhodopsin-regulated calcium
currents in Chlamydomonas. Nature 351, 489–491.
Harz, H., Nonnengässer, C., and Hegemann, P. (1992). The photoreceptor
current of the green alga Chlamydomonas. Phil. Trans. R. Soc.
London B Biol. Sci. 338, 39–52.
Hegemann, P., and Harz, H. (1998). How microalgae see the light. In
Microbial Responses to Light and Time. S. Caddick, D. Baumberg, A.
Hodgson, and M.K. Phillips-Jones, eds (Cambridge, UK: Cambridge
University Press), pp. 95–105.
Hofmann, K., and Stoffel, W. (1993). TMbase - A database of membrane
spanning proteins segments. Biol. Chem. Hoppe Seyler
374, 166.
Holland, E.M., Braun, F.J., Nonnengäßer, C., Harz, H., and
Hegemann, P. (1996). The nature of rhodopsin triggered photocurrents
in Chlamydomonas. I. Kinetics and influence of divalent cations.
Biophys. J. 70, 924–931.
Huang, K., and Beck, C.F. (2003). Phototropin is the blue-light receptor
that controls multiple steps in the sexual life cycle of the green alga
Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 100, 6269–
6274.
Huang, K., Kunkel, T., and Beck, C.F. (2004). Localization of the bluelight
receptor phototropin to the flagella of the green alga Chlamydomonas
reinhardtii. Mol. Biol. Cell 15, 3605–3614.

22 of 23 The Plant Cell
Huang, K., Merkle, T., and Beck, C.F. (2002). Isolation and characterization
of a Chlamydomonas gene that encodes a putative blue-light
photoreceptor of the phototropin family. Physiol. Plant 115, 613–622.
Johnson, C.H., Kondo, T., and Hastings, J.W. (1991). Action spectrum
for resetting the circadian phototaxis rhythm in the CW15 strains of
Chlamydomonas. II. Illuminated cells. Plant Physiol. 97, 1122–1129.
Kateriya, S., Nagel, G., Bamberg, E., and Hegemann, P. (2004).
‘‘Vision’’ in single-celled algae. News Physiol. Sci. 19, 133–137.
Keller, L.C., Romijn, E.P., Zamora, I., Yates, I.I.I., Jr., and Marshall,
W.F. (2005). Proteomic analysis of isolated Chlamydomonas centrioles
reveals orthologs of ciliary-disease genes. Curr. Biol. 15, 1090–1098.
Kessler, F., Schnell, D., and Blobel, G. (1999). Identification of proteins
associated with plastoglobules isolated from pea (Pisum sativum L.)
chloroplasts. Planta 208, 107–113.
Kindle, K.L. (1990). High-frequency nuclear transformation of Chlamydomonas
reinhardtii. Proc. Natl. Acad. Sci. USA 87, 1228–1232.
King, S.M. (1995). Large scale isolation of Chlamydomonas flagella.
Methods Cell Biol. 47, 9–12.
Kondo, T., Johnson, C.H., and Hastings, J.W. (1991). Action spectrum
for resetting the circadian phototaxis rhythm in the cw 15 strains of
Chlamydomonas. I. Cells in darkness. Plant Physiol. 95, 197–205.
Kreimer, G., Brohsonn, U., and Melkonian, M. (1991). Isolation and
partial characterization of the photoreceptive organelle for phototaxis
of a flagellate green alga. Eur. J. Cell Biol. 55, 318–327.
Kreimer, G., and Melkonian, M. (1990). Reflection confocal laser
scanning microscopy of eyespots in flagellate green alga. Eur. J.
Cell Biol. 53, 101–111.
Kreimer, G., Overländer, C., Sineshchekov, O.A., Stolzis, H.,
Nultsch, W., and Melkonian, M. (1992). Functional analysis of the
eyespot in Chlamydomonas reinhardtii mutant ey 627, mt ÿ . Planta
188, 513–521.
Kreimer, G. (1994). Cell biology of phototaxis in flagellated algae. Int.
Rev. Cytol. 148, 229–310.
Kreimer, G. (2001). Light reception and signal modulation during
photoorientation of flagellate algae. In Comprehensive Series in
Photosciences, M. Lebert and D.-P. Häder, eds (Amsterdam, The
Netherlands: Elsevier/North Holland), pp. 193–227.
Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E.L.L. (2001).
Predicting transmembrane protein topology with a hidden Markov
model: Application to complete genomes. J. Mol. Biol. 305, 567–580.
Lamb, M.R., Dutcher, S.K., Worley, C.K., and Dieckmann, C.L.
(1999). Eyespot assembly mutants in Chlamydomonas reinhardtii.
Genetics 153, 721–729.
Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: Pigments of
photosynthetic biomembranes. Methods Enzymol. 148, 350–382.
Linden, L., and Kreimer, G. (1995). Calcium modulates rapid protein
phosphorylation/dephosporylation in isolated eyespot apparatuses of
the green alga Spermatozopsis similis. Planta 197, 343–351.
Link, A.J., Eng, J., Schieltz, D.M., Carmack, E., Mize, G.J., Morris,
D.R., Garvik, B.M., and Yates III, J.R. (1999). Direct analysis of protein
complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.
Majeran, W., Cai, Y., Sun, Q., and van Wijk, K.J. (2005). Functional
differentiation of bundle sheath and mesophyll maize chloroplasts
determined by comparative proteomics. Plant Cell 17, 3111–3140.
Marmagne, A., Rouet, M.-A., Ferro, M., Rolland, N., Alcon, C.,
Joyard, J., Garin, J., Barbier-Brygoo, H., and Ephritikhine, G.
(2004). Identification of new intrinsic proteins in Arabidopsis plasma
membrane proteome. Mol. Cell. Proteomics 3, 675–691.
Melkonian, M., and Robenek, H. (1984). The eyespot apparatus of
green algae: A critical review. Prog. Phycol. Res. 3, 193–268.
Mergenhagen, D. (1984). Circadian clock: Genetic characterization of a
short period mutant of Chlamydomonas reinhardtii. Eur. J. Cell Biol.
33, 13–18.
Mittag, M. (1996). Conserved circadian elements in phylogenetically
diverse algae. Proc. Natl. Acad. Sci. USA 93, 14401–14404.
Mittag, M., Kiaulehn, S., and Johnson, C.H. (2005). The circadian
clock in Chlamydomonas reinhardtii. What is it for? What is it similar
to? Plant Physiol. 137, 399–409.
Morel-Laurens, N., and Feinleib, M.E. (1983). Photomovement in an
‘‘eyeless’’ mutant of Chlamydomonas. Photochem. Photobiol. 37,
189–194.
Nagel, G., Brauner, M., Liewald, J.F., Adeishvili, N., Bamberg, E.,
and Gottschalk, A. (2005). Light activation of channelrhodopsin-2 in
excitable cells of Caenorhabditis elegans triggers rapid behavioral
responses. Curr. Biol. 15, 2279–2284.
Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A.M.,
Bamberg, E., and Hegemann, P. (2002). Channelrhodopsin-1:
A light-gated proton channel in green algae. Science 296, 2395–
2398.
Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold,
P., Ollig, D., Hegemann, P., and Bamberg, E. (2003). Channelrhodopsin-2,
a directly light-gated cation-selective membrane channel.
Proc. Natl. Acad. Sci. USA 100, 13940–13945.
Nakamura, S., Ogihara, H., Jinbo, K., Tateishi, M., Takahashi, T.,
Yoshimura, K., Kubota, M., Watanabe, M., and Nakamura, S.
(2001). Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,
Chlorophyceae) mutant with multiple eyespots. Phycol. Res. 49,
115–121.
Neuhoff, V., Phillip, K., Zimmer, H.G., and Mesecke, S. (1979). A
simple versatile sensitive and volume-independent method for quantitative
protein determination which is independent of other external
influences. Hoppe Seylers Z. Physiol. Chem. 360, 1657–1670.
Nultsch, W. (1975). Phototaxis and photokinesis. In Primitive Sensory
and Communication Systems, M.J. Carlyle, ed (London, New York,
San Francisco: Academic Press), pp. 29–90.
Palczewski, K., and Saari, J.C. (1997). Activation and inactivation
steps in the visual transduction pathway. Curr. Opin. Neurobiol. 7,
500–504.
Panda, S., Hogenesch, J.B., and Kay, S.A. (2002). Circadian rhythms
from flies to human. Nature 417, 329–335.
Pazour, G., Sineshchekov, O.A., and Witman, G.B. (1995). Mutational
analysis of the phototransduction pathway of Chlamydomonas reinhardtii.
J. Cell Biol. 131, 427–440.
Pazour, G.J., Agrin, N., Leszyk, J., and Witman, G.B. (2005). Proteomic
analysis of a eukaryotic cilium. J. Cell Biol. 170, 103–113.
Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G.,
Rudella, A., Liberles, D.A., Soderberg, L., Roepstorff, P., von
Heijne, G., and van Wijk, K.J. (2002). Central functions of the luminal
and peripheral thylakoid proteome of Arabidopsis determined
by experimentation and genome-wide prediction. Plant Cell 14,
211–236.
Porter, M.E., and Sale, W.S. (2000). The 9þ2 axoneme anchors
multiple inner arm dyneins and a network of kinases and phosphatases
that control motility. J. Cell Biol. 151, F37–F42.
Pozueta-Romero, J., Rafia, F., Houlné, G., Cheniclet, C., Carde, J.P.,
Schantz, M.-L., and Schantz, R. (1997). A ubiquitous plant housekeeping
gene, PAP, encodes a major protein component of bell
pepper chromoplasts. Plant Physiol. 115, 1185–1194.
Preuss, F., Fan, J.Y., Kalive, M., Bao, S., Schuenemann, E., Bjes,
E.S., and Price, J.L. (2004). Drosophila doubletime mutations which
either shorten or lengthen the period of circadian rhythms decrease
the protein kinase activity of casein kinase I. Mol. Cell. Biol. 24,
886–898.
Rabilloud, T., Carpentier, G., and Tarroux, P. (1988). Improvement
and simplification of low-background silver staining by using sodium
dithionite. Electrophoresis 9, 288–291.

Reinders, J., Lewandrowski, U., Moebius, J., Wagner, Y., and
Sickmann, A. (2004). Challenges in mass spectrometry-based proteomics.
Proteomics 4, 3686–3703.
Renninger, S., Backendorf, E., and Kreimer, G. (2001). Subfractionation
of eyespot apparatuses from the green alga Spermatozopsis
similis: Isolation and characterization of eyespot globules. Planta 213,
51–63.
Renninger, S., Dieckmann, C.L., and Kreimer, G. (2006). Towards a
protein map of the green algal eyespot: Analysis of eyespot globuleassociated
proteins. Phycologia 45, 199–212.
Reppert, S.M., and Weaver, D.R. (2002). Coordination of circadian
timing in mammals. Nature 418, 935–941.
Rey, P., Gillet, B., Römer, S., Eymery, F., Massimino, J., Peltier, G.,
and Kuntz, M. (2000). Over-expression of a pepper plastid-lipidassociated
protein in tobacco leads to changes in plastid-ultrastructure
and plant development upon stress. Plant J. 21, 483–494.
Roberts, D.G.W., Lamb, M.R., and Dieckmann, C.L. (2001). Characterization
of the eye2 gene required for eyespot assembly in Chlamydomonas
reinhardtii. Genetics 158, 1037–1049.
Ruch, S., Beyer, P., Ernst, H.G., and Al-Babili, S. (2005). Retinal
biosynthesis in Eubacteria: In vitro characterization of a novel carotenoid
oxygenase from Synechocystis sp. PCC 6803. Mol. Microbiol.
55, 1015–1024.
Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press).
Sato, E., Sagami, I., Uchida, T., Sato, A., Kitagawa, T., Igarashi, J.,
and Shimizu, T. (2004). SOUL in mouse eyes is a new hexameric
heme-binding protein with characteristic optical absorption, resonance
raman spectral, and heme-binding properties. Biochemistry 43,
14189–14198.
Schaller, K., and Uhl, R. (1997). A microspectrophotometric study of
the shielding properties of eyespot and cell body in Chlamydomonas.
Biophys. J. 73, 1573–1578.
Schweighofer, A., Hirt, H., and Meskiene, I. (2004). Plant PP2C
phosphatases: Emerging functions in stress signaling. Trends Plant
Sci. 9, 236–243.
Shimada, H., Koizumi, M., Kuroki, K., Mochizuki, M., Fujimoto, H.,
Ohta, H., Masuda, T., and Takamiya, K.-I. (2004). ARC3, a chloroplast
division factor, is a chimera of prokaryotic FtsZ and part of a
eukaryotic phosphatidylinositol-4-phosphate 5-kinase. Plant Cell
Physiol. 45, 960–967.
Sineshchekov, O.A., and Govorunova, E.G. (1999). Rhodopsin-mediated
photosensing in green flagellated algae. Trends Plant Sci. 4, 58–63.
Sineshchekov, O.A., and Govorunova, E.A. (2001). Electrical events in
photomovement of green flagellated algae. In Comprehensive Series
in Photosciences, M. Lebert and D.-P. Häder, eds (Amsterdam, The
Netherlands: Elsevier/North Holland), pp. 245–280.
Sineshchekov, O.A., Jung, K.-H., and Spudich, J.L. (2002). Two rhodopsins
mediate phototaxis to low- and high-intensity light in Chlamydomonas
reinhardtii. Proc. Natl. Acad. Sci. USA 99, 8689–8694.
Sizova, I., Fuhrmann, M., and Hegemann, P. (2001). A Streptomyces
rimosus aphVIII gene coding for a new type phosphotransferase
provides stable antibiotic resistance to Chlamydomonas reinhardtii.
Gene 227, 221–229.
Chlamydomonas Eyespot Proteome 23 of 23
Suzuki, T., et al. (2003). Archael-type rhodopsins in Chlamydomonas:
Model structure and intracellular localization. Biochem. Biophys. Res.
Commun. 301, 711–717.
Szurmant, H., and Ordal, G.W. (2004). Diversity in Chemotaxis mechanisms
among bacteria and archea. Microbiol. Mol. Biol. Rev. 68,
301–319.
Takeshima, H., Komazaki, S., Nishi, M., Iino, M., and Kangawa, K.
(2000). Junctophilins: A novel family of junctional membrane complex
proteins. Mol. Cell 6, 11–22.
von Heijne, G. (1992). Membrane protein structure prediction: Hydrophobicity
analysis and the ‘positive inside’ rule. J. Mol. Biol. 225,
487–494.
Wagner, V., Fiedler, M., Markert, C., Hippler, M., and Mittag, M.
(2004). Functional proteomics of circadian expressed proteins from
Chlamydomonas reinhardtii. FEBS Lett. 559, 129–135.
Wagner, V., Geßner, G., Heiland, I., Kaminski, M., Hawat, S.,
Scheffler, K., and Mittag, M. (2006). Analysis of the phosphoproteome
of Chlamydomonas reinhardtii provides new insights into
various cellular pathways. Eukaryot. Cell 5, 457–468.
Walne, P.L., and Arnott, H.J. (1967). The comparative ultrastructure
and possible function of eyespots: Euglena granulata and Chlamydomonas
eugametos. Planta 77, 325–353.
Waltenberger, H., Schneid, C., Grosch, J.O., Bareiß, A., and Mittag,
M. (2001). Identification of target mRNAs from C. reinhardtii for
the clock-controlled RNA-binding protein Chlamy 1. Mol. Genet.
Genomics 265, 180–188.
Washburn, M.P., Wolters, D., and Yates III, J.R. (2001). Large-scale
analysis of the yeast proteome by multidimensional protein identification
technology. Nat. Biotechnol. 19, 242–247.
Webre, D.J., Wolanin, P.M., and Stock, J.B. (2003). Bacterial chemotaxis.
Curr. Biol. 13, R47–R49.
Wei, Z.M., Laby, R.J., Zumoff, C.H., Bauer, D.W., He, S.Y., Collmer,
A., and Beer, S.V. (1992). Harpin, elicitor of the hypersensitive
response produced by the plant pathogen Erwinia amylovora. Science
257, 85–88.
Witman, G.B. (1993). Chlamydomonas phototaxis. Trends Cell Biol. 3,
403–408.
Yamaguchi, K., Prieto, S., Beligni, M.V., Haynes, P.A., McDonald,
W.H., Yates III, J.R., and Mayfield, S.P. (2002). Proteomic characterization
of the small subunit of Chlamydomonas reinhardtii chloroplast
ribosome: Identification of a novel S1 domain-containing protein
and unusually large orthologs of bacterial S2, S3, and S5. Plant Cell
14, 2957–2974.
Yang, P., and Sale, W.S. (2000). Casein kinase I is anchored on
axonemal doublet microtubules and regulates flagellar dynein phosphorylation
and activity. J. Biol. Chem. 275, 18905–18912.
Zhao, B., Schneid, C., Iliev, D., Schmidt, E.-M., Wagner, V., Wollnik,
F., and Mittag, M. (2004). The circadian RNA-binding protein
CHLAMY 1 represents a novel type heteromer of RNA recognition
motif and lysine homology-containing subunits. Eukaryot. Cell 3,
815–825.
Zylka, M.J., and Reppert, S.M. (1999). Discovery of a putative hemebinding
protein family (SOUL/HBP) by two-tissue suppression subtractive
hybridization and database searches. Brain Res. Mol. Brain
Res. 74, 175–181.

Proteomic Analysis of the Eyespot of Chlamydomonas reinhardtii Provides Novel Insights into Its
Components and Tactic Movements
Melanie Schmidt, Gunther Geßner, Matthias Luff, Ines Heiland, Volker Wagner, Marc Kaminski, Stefan
Geimer, Nicole Eitzinger, Tobias Reißenweber, Olga Voytsekh, Monika Fiedler, Maria Mittag and
Georg Kreimer
Plant Cell;
originally published online June 23, 2006;
DOI 10.1105/tpc.106.041749
Supplemental Data
Permissions
eTOCs
CiteTrack Alerts
Subscription Information
This information is current as of December 23, 2012
http://www.plantcell.org/content/suppl/2006/06/23/tpc.106.041749.DC1.html
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY